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  • 1995-1999  (1,831)
  • 1995  (1,831)
  • Biochemistry and Biotechnology  (1,318)
  • Computational Chemistry and Molecular Modeling  (513)
  • Nuclear reactions
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  • 1995-1999  (1,831)
Year
  • 201
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 317-325 
    ISSN: 0952-3499
    Keywords: monoclonal antibody ; protein structure ; water structure ; x-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The solvation of the antibody-antigen Fv D1.3-lysozyme complex is investigated through a study of the conservation of water molecules in crystal structures of the wild-type Fv fragment of antibody D1.3, 5 free lysozyme, the wild-type Fv D1.3-lysozyme complex, 5 Fv D1.3 mutants complexed with lysozyme and the crystal structure of an idiotope (Fv D1.3)-abti-idiotope (Fv E5.2) complex. In all, there are 99 water molecules common to the wild-type and mutant antibody-lysozyme complexes. The antibody-lysozyme interface includes 25 well-ordered solvent molecules, conserved among the wild-type and mutant Fv D1.3-lysozyme complexes, which are bound directly or through other water molecules to both antibody and antigen. In addition to contributing hydrogen bonds to the antibody-antigen interaction the solvent molecules fill many interface cavities. Comparison with x-ray crystal structures of free Fv D1.3 and free lysozyme shows that 20 of these conserved interface waters in the complex were bound to one of the free proteins. Uo to 23 additional water molecules are also found in the antibody-antigen interface, however these waters do no bridge antibody and antigen and their temperature factors are much higher than those of the 25 well-ordered waters. Fifteen water molecules are displaced to form the complex, some of which are substituted by hydrophilic protein atoms, and 5 water molecules are added at the antibody-antigen interface with the formation of the complex. While the current crystal models of the D1.3-lysozyme complex do not demonstrate the increase in bound waters found in a physico-chemical study of the interaction at decreased water activities, the 25 well-ordered interface water contribute a net gain of 10 hydrogen bonds to complex stability.
    Additional Material: 6 Ill.
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  • 202
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 167-168 
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 203
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 197-210 
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 204
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 334-344 
    ISSN: 0952-3499
    Keywords: occluded surface ; protein packing ; misfolded protein Structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We describe a novel method to calculate the packing interactions in protein structural models. The method calculates the interatomic occluded surface areas for each atom in the protein model. The identification of, and degree of interaction with, neighboring atoms is accomplished by extending surface normal from a dot surface of each atom to the point of intersection with neighboring atoms. The combined occluded and non-occluded surface areas may be normalized for the amino acid composition of the protein providing a single parameter, the normalized protein surface ratio, which is diagnostic for native-like Structures. Individual residues in the model which are in infrequent occluded surface environments may be identified. The method provides a means to explicitly describe packing densities and packing environments of individual atoms in a protein model. Finally, the method allows estimation of the complementarity between any interacting molecules, for example a ligand binding to a receptor.
    Additional Material: 7 Ill.
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  • 205
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995) 
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 206
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 9-28 
    ISSN: 0952-3499
    Keywords: protein structure prediction ; molecular evolution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recent progress in structure prediction has allowed bona fide predictions, those made and published before an experimental structure is determined, to be remarkably accurate. The most successful methods rely on an analysis of patterns of conservation and variation within homologues protein sequences, extract tertiary structural information before secondary structure is predicted, and a avoid ‘three state per residue scores’ as a tool for evaluating a prediction, focusing instead on efforts to understand why a prediction is successful when it is successful, and why it fails when it fails.
    Additional Material: 13 Ill.
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  • 207
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 35-39 
    ISSN: 0952-3499
    Keywords: molecular imprinting ; surface imprinting ; separation/purification of proteins ; ribonuclease A ; metal coordination ; chelator ; surface-exposed histidines ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An Adsorbent showing enhanced selectivity for the enzyme RNase A was prepared by a surface imprinting procedure based on metal coordination. A metal chelating monomer, N-(4-vinyl)-benzyl iminodiacetic acid, was polymerized onto methacrylate-derivated silica particles in the presence of RNase A and metal ions. Lysozyme and RNase A were separated on the adsorbent used as stationary phase in high-performance liquid chromatography.
    Additional Material: 2 Ill.
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  • 208
    ISSN: 0952-3499
    Keywords: topological templates ; structural motif based library ; protein surface mimetic ; template-assembled side chains (TASK) ; de novo design ; TASP concept ; peptide library ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Topological templates disposing orthogonally protected reactive sites in defined spatial positions of a variable degree of flexibility can serve as a pool for the selective functionalizations by groups thought to interact with an acceptor molecule. This strategy is exemplified by the chemo-selective ligation of amino-acid residues to a cyclic peptide as a structural motif (template-assembled side chains; TASK) applying the concept of combinatorial libraries. Binding assays to a monoclonal antibody allow for the delineation of a lead compound mimicking the structural features of a discontinuous epitope. The conceptually novel approach may serve as a tool in molecular recognition studies and in drug development.
    Additional Material: 5 Ill.
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  • 209
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 47-51 
    ISSN: 0952-3499
    Keywords: photo-affinity albelling ; receptors ; melanocyte stimulating hormone (MSH) ; melanophore ; agonism ; antagonism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Photocrosslinking of receptor for α-melanocyte-stimulating hormone (α-MSH) on melanophores of frogs and lizards has been shown to induce long-lasting receptor stimulation whereby the photoreactive α-MSH may contain one or two photolables in positions 1, 7, 9, or 13. The chemical synthesis and biological testing of an α-MSH analogue is now described which contains three photoreactive groups in positions 1, 9 and 13, on eof which with a cleavable S—S disulphide bridge: [ApSSpr-Ser1, Trp(Naps)9 Pap13]-α-MSH. Photocrosslinking of MSH receptors on melanophores of Anolis carolinensis with this analogue led to almost complete receptor blockade which could be transformed into long-lasting receptor stimulation by exposure to a thiol reagents. By contrast, the analogue containing only two photoreactive groups in positions 9 and 13, [Trp(Naps)9, Pap13]-α-MSH, produced long-lasting receptor stimulation which was not altered by the thiol reagent. These results demonstrate that one and the same peptide ligand may contain structural information for both receptor activation and inhibition and that the receptor may become arrested in an activated or inhibited state by multiple photocrosslinking, depending on the relative positions of these crosslinks.
    Additional Material: 3 Ill.
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  • 210
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 59-62 
    ISSN: 0952-3499
    Keywords: drug targeting ; viral surface glycoproteins ; glycoproteins ; HIV-1 gp160 ; gene delivery ; ricin-a ; artificial viral envelopes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The goal of this study was to exploit molecular recognition of cell surface receptors by viral surface glycoproteins as a means for the selective intracellular delivery of macromolecules. To accomplish this, artificial viral envelopes (AVE) resembling the human immunodeficiency virus-1 (HIV-1) were designed as a model system. Recombinant HIV-1 surface glycoprotein gp160 (HIV-1 rgp 160) was inserted in the artificial envelope by a two-step detergent dialysis process. The artificial HIV-1 envelope recognized the CD4 cell surface receptor. FITC-dextran and ricin A were employed as model macromolecules as they cannot passively diffuse across cell membranes. Selective transfer of FITC-dextran encapsulated in HIV-1 rgp160 AVE into a CD4-positive cell line (REX-1B) versus a CD4-negative cell line (KG-1) was demonstrated. Ricin A at concentrations as low as 2 ng/ml arrested cell growth of CD4-positive MOLT-4 cells, whereas 8ng/ml ricin A in solution had no effect on cell growth. The arrest of cell growth was reverted in the presence of excess anti-gp120 monoclonal antibody. Naked enveloped (without HIV-1 rgp 160 inserted) were alsofound to interact with cells and transfer material, although less efficiently and in a non-specific manner. Viral mimicry using AVE may be a means for targeted intracellular delivery of peptides, proteins, enzymes, toxins, oligodeoxynucleotides, gene constructs, and other non-diffusive, labile or toxic macromolecules.
    Additional Material: 3 Ill.
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  • 211
    ISSN: 0952-3499
    Keywords: cytokine ; receptor ; kinetics ; biosensor ; interaction ; screening ; ELISA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring as ELSIA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5Rα-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor α subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of theFc chimera attached by oriented immobilization via protein A. Hence, shIL5Rα-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5Rα-Fc receptor complex. The binding was high affinity (Kdapp=6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5Rα. The results demonstrate a novel use of real-time biosensor technology as a macromolecular interaction analysis tool to help develop other assay using immobilized ligands, including high throughout screening assays to identify antagonist leads. The biosensor technology also offers a means to evaluate the mechanism of action of lead compounds by competition binding, as well as by direct binding if the leads are large enough or can be immobilized to the sensor surface.
    Additional Material: 6 Ill.
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  • 212
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 90-94 
    ISSN: 0952-3499
    Keywords: optimisation ; protein G chromatography ; monoclonal antibodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Compared to more stable chromatographic media the use of affinity media with biological ligands such as Protein G Sepharose® 4 Fast Flow poses special challenges regarding regeneration and sanitization. This is especially critical for the purification of pharmaceutical proteins, where complete regeneration of the column between runs is of paramount importance. Here, the problems encountered during process development and up scaling of regeneration methods for a protein G Sepharose Fast Flow column intended for the large-scale purification of pharmaceutical monoclonal anitbiodies are reported. The initially chosen alkaline regeneration buffer led to an increase in the affinity of Protein G towards antibodies which made elution increasingly difficult. A combination of urea and acetic acid was selected to ensure efficient cleaning of the matrix without affecting ligand properties.Validation experiments were done to demonstrate the functional integrity of the matrix after repeated cycles of use and regeneration, as well as the efficiency of the cleaning process.
    Additional Material: 7 Ill.
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  • 213
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 85-89 
    ISSN: 0952-3499
    Keywords: adsorption kinetics ; albumin ; monoclonal antibodies ; chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A chromatographic method was employed to study the kinetics of human serum albumin (HSA) adsorbed on immobilized monoclonal antibodies. The antibodies of various specificities were covalently bound to a high performance liquid chromatography (HPLC) silica support. For very low desorption rates, successive amounts of the reacting protein were injected until column saturation. The analysis of the increase of the non-retained fraction calculated from peak area measurements gives the capacity of the support and the rate of the biospecific adsorption process.The model is based on a second-order Langmuir kinetic law and assumes a global mass transfer for the adsorption process. The use of a silica support of small pore size permits the reduction of the contribution for mass transfer in the stagnant fluid and the decrease in the column capacity: due to its large size, the reacting molecule is adsorbed on the external surface of the particle.The adsorption rate constants of HSA on five monoclonal anti_HSA antibodies of different specificities were determined. For all the immuno-adsorbents studied, the adsorption rate constant is significantly lower than that found on immobilized polyclonal antibodies. Measurements at different flow rates reveal that the mass transfer due to the transport to the adsorbent surface is small and can be estimated.
    Additional Material: 3 Ill.
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  • 214
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 100-105 
    ISSN: 0952-3499
    Keywords: acidic glycans ; colon carcinomas ; cell adhesion ; embryogenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new class of acidic glycans isolated from marine sponges and sea urchin embryos was shown to mediate cell adhesion via carbohydrate-carbohydrate interactions. Cell aggregation blocking monoclonal antibodies (Block 1 and Block 2 mAbs) directed against these polysaccharides localized functional epitopes in embryonic sea urchin gut. Immunofluorescence light microscopy of human colon carcinomas and healthy colon samples with Block 1 and Block 2 mAbs established that the carbohydrate structures similar to the invertebrate acidic glycan adhesion molecules are also expressed in humans. The Block 1 mAb labeled basal and apical lamina of tumor cells, whereas the Block 2 bound exclusively to the apical part of the epithelium. In normal tissue whole goblet cell membrane was stained with both antibodies indicating that transformation leads to spatial rearrangement of glycan antigens. Acidic glycans from human colon carcinomas and normal colon were isolated after delipidation, and complete pronase and DNase digestion, by gel filtration and adsorption to anion exchange membranes. Immunodot assay with Block 1 and Block 2 mAbs revealed that tumor cells have elevated expression of both carbohydrate structures. These results suggest that the acidic glycan adhesion molecules, originally found in sponges and sea urchin embryos, may represent a new class of carbohydrate carcino-embryonal antigens involved in cellular interactions associated with morphogenesis, metastasis and renewal of adult tissue.
    Additional Material: 3 Ill.
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  • 215
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 116-124 
    ISSN: 0952-3499
    Keywords: cyanogen bromide ; 2,2,2-trifluoroethanesulfonyl chloride ; thiophilic interactions ; base-atom recognition ; calmodulin ; fibrinogen ; phosphorylase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The cyanogen bromide and tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) methods belong to the best-known activation procedures for solid supports in biochemistry. In both cases the originally proposed reaction mechanisms were revised many years later. In this paper important aspects of these two major activation reactions in connection with the singular polysaccharide support, agarose, will be treated with emphasis on the novel reaction mechanism recently proposed for tresyl chloride. In addition, the special role played by sulfur in the new uncharged alkyl-S-S-gels is examined in connection with the phenomenon of base atom recognition.
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  • 216
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 67-71 
    ISSN: 0952-3499
    Keywords: conformation ; calmodulin ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Calmodulin, similarly to many other Ca2+ -activated proteins, undergoes considerable conformational changes in the presence of Ca2+ ions. These changes were followed using specific monoclonal antibodies against calmodulin. Since calmodulin is a poor immunogen due to ties high phylogenetic conservancy, glutaraldehyde-crosslinked bovine brain extract, which contains a considerable amount of functionally active calmodulin complexed with its target proteins, was used as an antigen. Out of nine anti-calmodulin mAbs isolated, three (namely, CAM1, CAM2, and CAM4) were purified and characterized. MAb CAM1 was identified as an IgG1 while mAbs CAM2 and CAM4 belong to IgM class. Additively ELISA showed that mAb CAM1 binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize cloe epitopes. MAb CAM1 was found to be especially sensitive to the conformational state of calmodulin in the presence of Ca2+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca2+ removal. In addition mAb CAM1 failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM2 and CAM4 share common epitopes with the above molecules.
    Additional Material: 4 Ill.
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  • 217
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    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 72-76 
    ISSN: 0952-3499
    Keywords: refolding ; monoclonal antibodies ; carboxypeptidase A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The availability of monoclonal antibodies which bind to a specific antigen at distinct and well-defined sites has led to a better understanding of the effects of highly specific enzyme-antibody interactions on enzyme behaviour. By appropriate selection it has been possible to isolate those antibodies that are non-inhibitory to biological activity of the enzyme and bind at strategic locations on the antigen molecule, resulting in a considerable stabilization effect on the enzyme conformation. Moreover, such monoclonal antibodies proved to have a chaperone activity leading to aconsiderable refolding effect on the enzyme which was already partially heat denatured. Renaturation of carboxypeptidase A after heat denaturation in the presence of selected monoclonal antibodies, was followed by recovery of its enzymatic activity. The refolding effect of anti-CPA monoclonal antibodies, was followed by recovery of its enzymatic activity. The refolding effect of anit-CPA monoclonal antibodies on heat-denatured enzyme depends on the degree of denaturation of the enzyme and on the location of the antigenic site of each antibody. The additively effect of the pairs of monoclonal antibodies on the refolding process of CPA proved to be dependent on the localization of the antigenic sites of the monoclonal antibodies studied.
    Additional Material: 5 Ill.
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  • 218
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 77-84 
    ISSN: 0952-3499
    Keywords: metal ion affinity ; aqueous two-phase partition ; normal and pathological human cells ; primary ; secondary cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Immobilized metal-ion affinity partitioning (IMAP) is shown to be useful as a preliminary screening test and for the separation of different cell populations based upon recognition of the differences in the proteins on cell surfaces. The feasibility of using IMAP to segregate a spectrum of normal human cells (red blood cells, lymphocytes and fibroblasts) from their counterpart pathological cells has been demonstrated.A clear segregation between normal and sickle-cell anemia red blood cells (RBC), or malaria (Plasmodium vivax) infected RBCs was obtained. Further, the partition differences were found to depend on the nature and the concentration of metal used.Cells form breast cancer and those from the lugn adenocarcinoma showed differences in their partition pattern as compared to normal fibroblasts when PEG-IDA-M(II) was added to the phase system. Maximum differences between the three cell populations were observed in the presence of 10% PEG-IDA-Ni(II).Normal lymphocytes and Burkitt's lymphoma cells (Raji nad Namalwa cell lines) were shown to partition differently in the presence of PEG-IDA-M(II) in the phase system. Normal lymphocytes could be distinguished from the Burkitt's lymphoma cell lines in all three phases (top, interface and bottom), in the presence of 10% PEG-IDA-Ni(II) in the system.These differences in the partition behavior could mainly be attributed to the density, surface exposure and micro-environment of histidine residues of cell membrane-associated proteins. These data, along with those obtained for normal and pathological human cells show that IMAP could be a simple and versatile tool for the segregation and study of cells.
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  • 219
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    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 8 (1995), S. 106-110 
    ISSN: 0952-3499
    Keywords: hemoglobin ; sugar ; recognition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Hemoglobin (Hb) is not a glycoprotein but can easily by glycosylated by a non-enzymatic mechanism. Based on some kinetic particularities, consisting in a faster glycosylation of the amine terminal group of the β-chain located on the allosteric site, a hypothesis of a recognition center for specific sugars has been advanced.Affinity chromatography materials based on epichlorohydrin crosslinked amylose, agarose and dextran (Sephadex®), were used. A specific interaction with the CL-amylose (α-1,4-glycosidic links) was found, while for Sephadex® (α-1,6-glycosidic links), no Hb retention was observed. An affinity retention of hemoglobins (human and bovine) on agarose (a galactose containing carbohydrate) has also been established.Chromatographic studies of Hb competitive elution with several monosaccharides (glucose, fructose, galactose) and disaccharides (saccharose, cellobiose, maltose, lactose) indicated that Hb retained by affinity onto CL-Amylose columns, can be eluted only with galactose and lactose; with the other tested saccharides, very poor or no desorption at all, has been observed. These new data suggest: (1) a selective recognition of a limited number of (poly)saccharidic materials; and (2) a different behavior towards various sugars, the best interaction being observed with galactose-containing sugars (lactose). These aspects fit well with the hypothesis of a hemoglobin recognition center for sugars.
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  • 220
    ISSN: 0952-3499
    Keywords: dextran matrix ; antibody immobilization ; antibody binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The authors have recently described the development of a carboxymethyl dextran-based sensor surface for biospecific interaction analysis by surface plasmon resonance. Ligands are immobilized via primary amine groups after activation of the carboxymethyl groups on the sensor surface with a mixture of N-hydroxysuccinimide and N-ethyl-N′-(dimethylaminopropyl) carbodiimide. Methods have now been developed for efficient immobilization via thiol/disulfide exchange, aldehyde coupling and biotin-avidin coupling. The specific activity of monoclonal antibodies immobilized by the four different methods was investigated by altering the immobilization conditions, e. g., activation time, protein concentration, ionic strength and the degree of modification, etc. Investigations have also been made concerning possible differences in the specific activity for antibodies immobilized using optimized conditions with respect to the four different chemistries. These studies show that, with the flexible carboxymethyl dextran matrix used here, the immobilization methods give rise to only minor differences in specific activity. Thus, with this solid support, a ‘site directed’ immobilization strategy for monoclonal antibodies has no advantage. In general the specific activity for optimized systems was approximately 75% for the binding of β2μ-globulin to an immobilized monoclonal antibody directed against β2μ-globulin. Reduced specific activities of immobilized antibodies induced by variation of the coupling conditions could be attributed to the deterioraton of the active site of the antibody.
    Additional Material: 7 Ill.
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  • 221
    ISSN: 0952-3499
    Keywords: association rate affinity ; biospecific interaction analysis ; dissociation rate ; Kinetics ; protein engineering ; staphylococcal protein A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A real time biospecific interaction analysis (BIA) was performed to study the specific interaction between the Fc portion of human immunoglobulin G1 (Fc1) and a one domain analogue (designated Z) of staphylococcal protein A, in monovalent (Z) and divalent (ZZ) forms, and five different single amino acid substituted Z variants (L17D, N28A, F30A, I31A, K35A). Experimental BIA data were used to calculate association rate constants (Kon), dissociation rate constants (koff). The divalent form (ZZ) showed a higher affinity for Fc1 mainly because of a slower off rate. Out of the five mutant Z protein, four (L17D, N28A, I31A, K35A) four had the major effect to Fc1 compared to the parent Z molecule. Surprisingly, two (L17D, I31A) of these four had the major effect of a decreased binding energy as a lowered kon while the other two (N28A, K35A) mutant proteins showed an increased koff as the major kinetic difference from Z in their binding to Fc1. For five of the six different Z variants, as well as for the ZZ molecule, calculated kaff and calculated differences in binding free energies relative to the parent Z molecule (ΔΔG), are in good agreement with the corresponding values obtained in a competitive displacement assay using radioactively labeled Z as a tracer (Cedergren et al., (1993) Prot. Eng. 6, 441-448). However, the I31A variant, with a measured kon that was more than three orders of magnitude lower than that of Z in the BIA assay, showed a significant weaker affinity to FcI when calculated from BIA data compared ot the competitive displacement assay. The discrepancy between these two methods for Z(I31A) is discussed as well as possible explanations for the unexpected large effect of lowered kon for two of the mutant Z proteins.
    Additional Material: 6 Ill.
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  • 222
    ISSN: 0952-3499
    Keywords: human and simian immunodeficiency viruses ; CD4 cell receptor ; acid anhydrides ; monoclonal antibodies ; serum albumin ; casein ; milk ; prophylaxis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The chemical transformation of synthetic combinatorial libraries to increase the diversity of compounds of medicinal interest was reported recently. Chemical modification of natural products represents a complementary approach to accomplish this aim. Modification of lysines by aromatic acid anhydrides, preferentially by 3-hydroxyphthalic and trimellitic anhydrides and trimellitic anhydride chloride, converted commonly available proteins (human and bovine serum albumin and casein) into potent inhibitors of (i) binding between the HIV-1 gp120 envelope glycoprotein and the CD4 cell receptor, probably owing to their binding to CD4, and (ii) infection by HIV-1. Modified bovind milk proteins are also potent HIV-1 inhibitors and may have protential for anti-Hiv-1 prophylaxis.
    Additional Material: 11 Ill.
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  • 223
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 452-460 
    ISSN: 0006-3592
    Keywords: chromatography ; displacement ; protein purification ; cation exchange systems ; amino acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. © 1995 John Wiley & Sons, Inc.
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  • 224
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    Biotechnology and Bioengineering 48 (1995), S. 461-475 
    ISSN: 0006-3592
    Keywords: countercurrent gradient chromatography ; protein separation ; human serum albumin ; chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (〉2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. © 1995 John Wiley & Sons, Inc.
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  • 225
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    Biotechnology and Bioengineering 48 (1995), S. 481-489 
    ISSN: 0006-3592
    Keywords: bioseparation ; protein refolding ; reverse micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The recombinant production of proteins leads to inclusion bodies which contain aggregated proteins in active, partially active, and inactive conformational states. These aggregated proteins must be extracted from the inclusion bodies, unfolded, and carefully refolded to the active and the stable conformational state. Mechanistic models for protein refolding are briefly presented. Different strategies and protocols are presented that lead to the active and stable protein conformational state. The techniques presented include chaperonin-assisted refolding, amino acid substitution, polyethylene glycolassisted refolding, protein refolding in reverse micelles, and antibody-assisted refolding of proteins. The techniques presented together provide a reasonable framework of the state-of-the-art and may be carefully applied to the bioseparation of other proteins and biological macromolecules of interest. © 1995 John Wiley & Sons, Inc.
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  • 226
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    Biotechnology and Bioengineering 48 (1995), S. 520-528 
    ISSN: 0006-3592
    Keywords: human growth hormone ; animal cell culture ; purification ; serum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kilodaltons. With the aid of computer molecular simulation, an hGH analog was created by altering an hGH gene to reflect the change of one amino acid (glycine [G] 120 to arginine [R]) within the third α-helix of the hGH molecule. This hGH analog, named hGHG120R, was found to be an hGH antagonist. It may have important implications in treating human conditions in which hGH levels are abnormally high, as found in type I diabetics. Several hundred milligrams of purified hGHG120R were needed to determine the biological activity of the antagonist in animal models. A multistep downstream process was developed to purify hGHG120R from cultured mouse L cells transfected with the hGHG120R gene. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, reversed phase high performance liquid chromatography, phase separation, and lyophiliation. This work discusses the rationale for the design of the process and experimental results on the purification of hGHG120R using the process. © 1995 John Wiley & Sons, Inc.
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  • 227
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    Biotechnology and Bioengineering 48 (1995), S. 557-557 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 228
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    Biotechnology and Bioengineering 48 (1995), S. 573-584 
    ISSN: 0006-3592
    Keywords: state estimation ; structured modeling ; lipase ; Candida rugosa ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple structured mathematical model coupled with a methodology of state and parameter estimation is developed for lipase production by Candida rugosa in batch fermentation. The model describes the system according to the following qualitative observations and hypothesis: Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell where it is consumed, transformed, and stored. Lipase is excreted to the medium where it is distributed between the available oil-water interphase and aqueous phase. Cell growth is modulated by the intracellular substrate concentration. Model parameters have been determined and the whole model validated against experiments not used in their determination. The estimation problem consists in the estimation of three state variables (biomass, intra- and extracellular substrate) and two kinetic parameters by using only the on-line measurement provided by exhaust gas analysis. The presented estimation strategy divides the complex problem into three subproblems that can be solved by stable algorithms. The estimation of biomass (X) and the specific growth rate (μ), is achieved by a recursive prediction error algorithm using the on-line measurement of the carbon dioxide evolution rate. μ is then used to perform an estimation of intracellular substrate and the other kinetic parameter related to substrate transport (A) by an adaptive observer. Extracellular substrate is then evaluated by means of the estimated values of intracellular substrate and biomass through the material balance of the reactor. Simulation and experimental tests showed good performance of the developed estimator, which appears suitable to be used for process control and monitoring. © 1995 John Wiley & Sons, Inc.
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  • 229
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    Biotechnology and Bioengineering 48 (1995), S. 585-591 
    ISSN: 0006-3592
    Keywords: phenoloxidases ; enzyme immobilization ; reverse micelles ; organic gels ; biotic detoxification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4°C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. © 1995 John Wiley & Sons, Inc.
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  • 230
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    Biotechnology and Bioengineering 48 (1995), S. 737-744 
    ISSN: 0006-3592
    Keywords: biofilm ; mass transfer coefficient ; microelectrode ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Local mass transfer rates for an electrochemically formed microsink in an aerobic biofilm was measured by a mobile microelectrode using limiting current technique. Mass transfer coefficients varied both horizontally and vertically in the biofilm. The results implied the existence of an irregular biofilm structure consisting of microbial cell clusters surrounded by tortuous water channels. An unexpected increase of the local mass transfer coefficient just above the biofilm surface suggested the existence, of local flow instability in this region. As expected, the influence of bulk flow velocity on the local mass transfer rate decreased with increasing depth into the biofilm. Mass transfer coefficients fluctuated significantly inside microbial cell clusters, suggesting the existence of internal channels through which liquid could flow. A new conceptual model of biofilm microbial cluster structure is proposed to account for such biofilm microstructure irregularities. © 1995 John Wiley & Sons, Inc.
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  • 231
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    Biotechnology and Bioengineering 46 (1995), S. 258-269 
    ISSN: 0006-3592
    Keywords: biofilm ; detachment ; abrasion ; breakage ; airlift reactor ; hydrodynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In three-phase internal loop airlift reactors, the detachment of biomass from suspended biofilm pellets in the presence of bare carrier particles was investigated under nongrowth conditions. The detachment rate was dominated by collisions between bare carrier particles and biofilm pellets. The concentration of bare carrier particles and the carrier roughness strongly influenced the detachment rate. A change in flow regime from bubbling to slug flow considerably increased the detachment rate. Otherwise, the superficial gas velocity did not directly affect the detachment rate. The influence of particle size was not clear. The bottom clearance did not affect the detachment rate within the tested range. Other aspects of reactor geometry might be important. The main detachment processes were abrasion and breakage of biofilm pellets. During the detachment process, two phases could be distinguished. In the first phase the detachment was relatively high, and both breakage and abrasion of biofilm pellets occurred. During the second phase, breakage dominated and the detachment rate was lower. The two-phase behavior is explained by differences in strength between the inner and outer biofilm layers, possibly caused by variations in local growth rates during biofilm formation. Differences in growth history might also explain the various detachment rates observed with different biofilm batches. © 1995 John Wiley & Sons, Inc.
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  • 232
    ISSN: 0006-3592
    Keywords: D-lactate ; biosensor ; on-line monitoring ; fermentation ; carbon paste electrode ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production of D-lactic acid by Lactobacillus delbrueckii (ATCC 9649) during fermentation was monitored on-line with a reagentless D-lactate dehydrogenase modified carbon paste electrode in a flow injection system integrated with a filtration sampling device. The time delay between sampling and detection was approximately 6 min. The use of an electropolymerized ortho-phenylenediamine membrane on the elctrode resulted in a very selective sensor response with acceptable stability and sensitivity. The D-lactate concentrations determined on-line agreed well with those determined by a standard method, suggesting that this sensor system is suitable for on-line monitoring of fermentation processes. © 1995 John Wiley & Sons, Inc.
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  • 233
    ISSN: 0006-3592
    Keywords: D-lactate ; D-lactate dehydrogenase ; carbon paste electrode ; redox polymer ; biosensor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A reagentless carbon paste electrode was designed for D-lactic acid analysis in a flow injection system for the monitoring of the production of D-lactate in a batch fermentation. D-Lactate dehydrogenase, nicotinamide adenine dinucleotide (NAD+), a synthetic redox polymer containing covalently attached toluidine blue O as mediator, graphite powder, and paraffin oil were used for the construction of the modified carbon paste electrode. D-Lactate selectivity was indicated by insignificant responses from a variety of possible interfernces including L-lactate. The electrodes gave a linear response in the range between 0.05 and 5 mM D-lactate, with a detecting limit of 30 μM, allowing a sample throughput of 20 h-1. Preliminary investigations were made by covering the electrode surface with electropolymerized membranes. Satisfactory stability was observed, indicated by a reproducibility of 3.3% relative standard deviation (RSD, n = 31), with a non-membrane-covered electrode for the analysis of D-lactate in fermentation broth. A long-term stability (230 broth samples) was proven, suggesting the electrodes to have a good potential for use in on-line monitoring of fermentation processes. © 1995 John Wiley & Sons, Inc.
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  • 234
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    Biotechnology and Bioengineering 46 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 235
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    Biotechnology and Bioengineering 46 (1995), S. 285-290 
    ISSN: 0006-3592
    Keywords: yeast ; ethanol ; amylases ; strain development ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A procedure was developed for construction of industrial strains of distiller's yeast (Saccharomyces cerevisiae). It includes several steps: construction of congenic genetically marked haploid strains of opposite mating types starting from an industrial strain of hybrid nature, integrative transformation of the above haploid strains with a DNA fragment containing an expression cassette responsible for new technological facilities, and hybridization of transformants and isolation of final industrial homozygous strains under experimental conditions simulating commercial fermentation processes. This strategy permits the generation of strains that have desirable characteristics of traditional races of distiller's yeast along with new technological facilities determined by the particular expression cassette. Using this procedure, we have constructed an industrial strain with improved amylolytic activity. © 1995 John Wiley & Sons, Inc.
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  • 236
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    Biotechnology and Bioengineering 46 (1995), S. 291-297 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration-dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. © 1995 John Wiley & Sons, Inc.
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  • 237
    ISSN: 0006-3592
    Keywords: cartilage ; polyglycolic acid ; chondrocytes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Rabbit articular chondrocytes were seeded onto three-dimensional polyglycolic acid (PGA) scaffolds and placed into a closed bioreactor system. After 4 weeks of growth, meshes were examined for cartilage formation. Gross examination revealed solid, glistening material that had the appearance of cartilaginous tissue. Histologic examination revealed cell growth and deposition of extracellular matrix throughout the mesh with a less dense central core. Alcian blue and Safranin 0 staining showed deposition of glycosaminoglycans (GAGs). Immunostaining showed positive reactivity for type II collagen and chondroitin sulfate and no reactivity for type I collagen. Biochemical analysis showed collagen and GAG values to be 15% and 25% dry weight, respectively. Our results indicate that this type of system compares well with those previously described and should be useful for producing cartilage for evaluation in a clinical setting. © 1995 John Wiley & Sons, Inc.
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  • 238
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    Biotechnology and Bioengineering 46 (1995), S. 306-313 
    ISSN: 0006-3592
    Keywords: tissue engineering ; chondrocyte ; polymer scaffold ; microgravity bioreactor ; rotating vessel ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Tissue-engineered cartilage was cultivated under conditions of simulated microgravity using rotating bioreactors. Rotation randomized the effects of gravity on inoculated cells (chondrocytes) and permitted their attachment to three-dimensional (3D) synthetic, biodegradable polymer scaffolds that were freely suspended within the vessel. After 1 week of cultivation, the cells regenerated a cartilaginous extracellular matrix (ECM) consisting of glycosaminoglycan (GAG) and collagen types I and II. Tissue constructs grown in simulated microgravity had higher GAG contents and thinner outer capsules than control constructs grown in turbulent spinner flasks. Two fluid dynamic regimes of simulated microgravity were identified, depending on the vessel rotation speed: (i) a settling regime in which the constructs were maintained in a state of continuous free-fall close to a stationary point within the vessel and (ii) an orbiting regime in which the constructs orbited around the vessel spin axis. In the settling regime, the numerically calculated relative fluid-construct velocity was comparable to the experimentally measured construct settling velocity (2-3 cm/s). A simple mathematical model was used in conjunction with measured construct physical properties to determine the hydrodynamic drag force and to estimate the hydrodynamic stress at the construct surface (1.5 dyn/cm2). Rotating bioreactors thus provide a powerful research tool for cultivating tissue-engineered cartilage and studying 3D tissue morphogenesis under well-defined fluid dynamic conditions. © 1995 John Wiley & Sons, Inc.
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  • 239
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    Biotechnology and Bioengineering 46 (1995), S. 325-332 
    ISSN: 0006-3592
    Keywords: cell recycle ; fed-batch ; oxygen uptake ; dissolved oxygen ; Candida lipolytica ; citric acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed-batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h-1, a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L · h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell · h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L · h) were obtained in the fed-batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed-batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed-batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed-batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed-batch fermentor were dependent on the cycle time and dissolved oxygen concentration. © 1995 John Wiley & Sons, Inc.
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  • 240
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    Biotechnology and Bioengineering 46 (1995), S. 333-342 
    ISSN: 0006-3592
    Keywords: biodegradation ; chlorinated hydrocarbons ; trichloroethylene ; microbial kinetics ; chemostat ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Microbial degradation of trichloroethylene (TCE) has been demonstrated under aerobic conditions with propane. The primary objective of this research was to evaluate the feasibility of introducing a vapor phase form of TCE in the presence of propane to batch bioreactors containing a liquid phase suspension of Mycobacterium vaccae JOB5 to accomplish degradation. The reactor system consisted of three phases: a vapor phase introducing air, propane, and TCE; a liquid phase of the microbial suspension; and a solid phase in the form of the microorganisms. Long-term and initial rate experiments were conducted on three culture sets to evaluate microbial response. In two long-term test fed propane and approximately 0.1 mg/L and 1 mg/L of TCE, respectively, propane utilization was more efficient at the high TCE concentration (600 mmol propane/mmol TCE versus 11,900 mmol propane/mmol TCE), because the propane degradation rate was approximately the same for both tests (6.73 mg/L · h and 7.85 mg/L · h for the high and low tests). In addition, TCE utilization decreased after complete propane consumption. Initial rate tests on culture sets fed propane only revealed that cells with a history of exposure to a high concentration of TCE had the highest specific growth rate, but the lowest half-saturation constant (7.60e-3 h-1 and 0.10 mg/L, respectively). Tests fed variable TCE concentrations (0.031 to 5.378 mg/L in the liquid phase) with no propane showed TCE depletion but no biomass growth. The tests revealed that the TCE removal increased as the TCE concentration increased, indicating a greater removal efficiency at the higher concentrations. Tests with a constant initial propane concentration and variable liquid phase TCE concentration revealed that specific propane utilization was essentially the same. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 46 (1995), S. 343-350 
    ISSN: 0006-3592
    Keywords: reverse micelles ; decanol ; amino acid extraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The concentrations of dioctyldimethyl ammonium chloride (DODMAC) and 1-decanol in isooctane needed to form reverse micelles by phase contact have been determined. The behavior of these reverse micelles in the extraction of aspartic acid, glutamic acid, and threonine was studied by analyzing all of the ionic species in the aqueous phase. The amino acid is extracted from the aqueous phase by exchanging with the Cl- counterions of DODMAC in the reverse micelles. The ionic species in the reverse micelles tend toward their undissociated states as the water uptake by the reverse micelles decreases. The effect of 1-decanol on the extraction of the amino acids with two negative charges is due to the change in the water uptake of the reverse micelles. The concentration of DODMAC has no effect on the ion exchange of the amino acid with one negative charge with the Cl- counterions of DODMAC in the reverse micelles. Higher molar ratios of decanol to DODMAC favor the selective separation of amino acids with different charge numbers. © 1995 John Wiley & Sons, Inc.
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  • 242
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    Biotechnology and Bioengineering 46 (1995), S. 314-324 
    ISSN: 0006-3592
    Keywords: product formation ; kinetic model ; microbial cells ; mammalian cells ; substrate excess ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. © 1995 John Wiley & Sons, Inc.
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  • 243
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    Biotechnology and Bioengineering 46 (1995), S. 351-360 
    ISSN: 0006-3592
    Keywords: aggregates ; BHK ; hydrodynamics ; Kolmogoroff's microscale theory of turbulence ; size control ; stirred vessels ; kidney cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Baby hamster kidney (BHK) cell aggregates grown in stirred vessels with different working volumes and impeller sizes were characterized. Using batch cultures, the range of agitation rates studied (25-100 rpm) led to aggregates with maximum sizes of 150 μm. Necrotic centers were not observed and cell specific productivity was independent of aggregate size. High cell viability was found for both single and adherent cells without an increase in cell death when agitation rate was increased. The increase in agitation rate affected aggregates by reducing their size and increasing their concentration and cell concentration in aggregates, while increasing the fraction of free cells in suspension. The experimental relationship between aggregate size and power dissipation rate per unit of mass was close to -1/4, suggesting a correlation with a critical turbulence microscale; this was independent of vessel scale and impeller geometry over the range investigated. Viscous stresses in the viscous dissipation subrange (below Kolmogoroff eddies) appear to be responsible for aggregate breakage. Under intense agitation BHK cells grown in the absence of microcarriers existed as aggregates without cell damage, whereas cells grown on the surface of microcarriers were largely reduced. This is a clear advantage for scaleup purposes if aggregates are used as a natural immobilization system in stirred vessels. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 46 (1995), S. 371-374 
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; cell mass sensor ; optical density probe ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the highly competitive market of commercial bakers' yeast, fermentations are operated for maximum efficiency and minimum production cost. In order to maintain competitiveness, the fermentations must be highly consistent with minimum variation in yeast performance, maximum yield on raw materials, and minimum production of undesirable side products. The use of advanced instrumentation is of critical importance to achieving these goals by the production engineer. An in situ optical density probe was used to determine the yeast cell density in full-scale commercial bakers' yeast fermentations. The optical density probe results were compared with oxygen uptake rate analyses, packed cell volume, and off-line measured cell dry weights. The most accurate measurement of cell density was found to be the optical density probe. This instrument allowed the on-line determination of cell density with highly consistent results from fermentation batch to batch and with out the need for intermittent recalibration. © 1995 John Wiley & Sons, Inc.
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  • 245
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during a condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. This limitation can be relieved by (i) the recycling of pyruvate from PEP using PEP synthase (Pps) or (ii) use of non-PTS sugars such as xylose. Previous studies have shown the usefulness of overexpressing tktA (encoding transketolase), aroG, and pps (PEP synthase) for DAHP production in an aroB strain unable to utilize DAHP further. In the present study we confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters. In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, as predicted by the stoichiometric analysis. However, this Pps effect is observed only in the presence of both increased AroG and TktA. In xylose mimimal medium, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown here are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.
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  • 246
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    Biotechnology and Bioengineering 46 (1995), S. 388-392 
    ISSN: 0006-3592
    Keywords: volumetric mass transfer coefficient ; oxygen uptake rate ; probe response time ; dynamic gas out-gas in method ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: There are many dynamic methods for measuring the volumetric mass transfer coefficient. The “gas out-gas in” method can directly determine the volumetric mass transfer coefficient in a bioreactor system and provide estimates of the volumetric microbial oxygen uptake rate and the average oxygen saturation concentration at the gas-liquid interface. The errors on these parameters are large if the dissolved oxygen probe response time is not considered. For reliable measurements, deconvolution of the oxygen probe measurements must be made. © 1995 John Wiley & Sons, Inc.
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  • 247
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    Biotechnology and Bioengineering 46 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 248
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    Biotechnology and Bioengineering 46 (1995), S. 393-395 
    ISSN: 0006-3592
    Keywords: lipase-catalyzed esterification ; n-butyl oleate ; pervaporation ; membrane separation ; cellulose acetate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Esterification of oleic acid with n-butanol in the presence of Lipozyme® was carried out at 25°C in isooctane with various initial water activities. Initial reaction rate as well as equilibrium conversion decreased at high initial water activity. Therefore, removal of water present in the reaction mixtures was essential. A pervaporation process was applied to the lipase-catalyzed synthesis of n-butyloleate to remove water. Pervaporation selectively separated water from the reaction mixture using a nonporous polymeric membrane, cellulose acetate. Therefore, pervaporation is potentially applicable to remove the water produced from various enzymatic processes, such as synthesis of various esters, peptides, and glycosides in a solvent system as well as in a solvent-free system. © 1995 John Wiley & Sons, Inc.
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  • 249
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    Biotechnology and Bioengineering 46 (1995), S. 610-620 
    ISSN: 0006-3592
    Keywords: enzymes ; organic solvents ; supercritical fluids ; carbon dioxide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have previously demonstrated that the activity of the lipase (Candida cylindracea) catalyzed transesterification reaction between methylmethacrylate and 2-ethylhexanol in supercritical carbon dioxide is comparatively low. In this article, we have investigated the same reaction in supercritical carbon dioxide with a special emphasis on determining the extent of any interaction between the enzyme and carbon dioxide. Transesterification reaction rates in hexane and supercritical carbon dioxide are compared at different temperatures. In supercritical carbon dioxide, temperature was found to have no significant effect on reaction rate in the range of 40° to 55°C. Above 55°C, however, the reaction rate increased significantly as a function of temperature. It appears that carbon dioxide forms reversible complexes with the free amine groups on the surface of the enzyme. Direct evidence of modification was obtained using mass spectroscopy to detect the extent of modification of a pure protein. The kinetics of the reaction have been studied in hexane, and they obey a ping-pong bi-bi mechanism with inhibition by 2-ethylhexanol. The effect of bubbling carbon dioxide and/or fluoroform on the reaction rate in hexane at different temperatures suggests that the enzyme undergoes shear inactivation in hexane. © 1995 John Wiley & Sons, Inc.
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  • 250
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    Biotechnology and Bioengineering 47 (1995), S. 60-70 
    ISSN: 0006-3592
    Keywords: enzymes ; organic solvents ; lipase ; reverse micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the “anhydrous” enzyme suspension approach. © 1995 John Wiley & Sons, Inc.
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  • 251
    ISSN: 0006-3592
    Keywords: kinetic parameters ; organic solvents ; thermodynamic activities ; solvation ; lipases ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: When it is assumed that organic solvents do not interfere with the binding process nor with the catalytic mechanism, the contribution of substrate-solvent interactions to enzyme kinetics can be accounted for by just replacing substrate concentrations in the equations by thermodynamic activities. It appears from the transformation that only the affinity parameters (Km, Ksp) are affected by this. Thus, in theory, the values of these corrected, intrinsic parameters (Kmint, kspint) and the maximal rate (V1) should be equal for all media. This was tested for hydrolysis, transesterification, and esterification reactions catalyzed by pig pancreas lipase and Pseudomonas cepacia lipase in various organic solvents. Correction was carried out via experimentally determined activity coefficients for the substrates in these solvents or, if not feasible, from values in data bases. However, although the kinetic performances of each enzyme in the solvents became much more similar after correction, differences still remained. Analysis of the enzyme suspensions revealed massive particles, which explains the low activity of enzymes in organic solvents. However, no correlation was found between estimates of the amount of catalytically available enzyme (present at the surface of suspended particles or immobilized on beads) and the maximal rates observed. Moreover, the solvents had similar effects on the intrinsic parameters of suspended and immobilized enzyme. The possible causes for the effects of the solvents on the catalytic performance of the enzymes, remaining after correction for solvent-substrate interactions and the amount of participating enzyme, are discussed with respect to the premises on which the correction method is based. © 1995 John Wiley & Sons, Inc.
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  • 252
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    Biotechnology and Bioengineering 48 (1995), S. 490-500 
    ISSN: 0006-3592
    Keywords: protein ; peptide ; oxidation ; metal catalysis ; photooxidation ; chelator ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Oxidation is one of the major chemical degradation pathways for protein pharmaceuticals. Methionine, cysteine, histidine, tryptophan, and tyrosine are the amino acid residues most susceptible to oxidation due to their high reactivity with various reactive oxygen species. Oxidation during protein processing and storage can be induced by contaminating oxidants, catalyzed by the presence of transition metal ions and induced by light. Oxidative modification depends on the structural features of the proteins as well as the particular oxidation mechanisms inherent in various oxidative species, and may also be influenced by pH, temperature, and buffer composition. Protein oxidation may result in loss of biological activity and other undesirable pharmaceutical consequences. Strategies to stabilize proteins against oxidation can be classified into intrinsic methods (site-directed mutagenesis and chemical modification), physical methods (solid vs. liquid formulations) and use of chemical additives. The optimum choice of chemical additives needs to be evaluated on the basis of the specific oxidation mechanism. Oxidation induced by the presence of oxidants in the system is referred to as a non-site-specific mechanism. Under such conditions, oxidation can be effectively inhibited by the appropriate addition of antioxidants or free radical scavengers. metal-catalyzed oxidation is a site-specific process, in which the addition of antioxidants may accelerate the oxidation reaction. Careful screening of chelating agents has been shown to be an alternative method for preventing metal-catalyzed oxidation. © 1995 John Wiley & Sons, Inc.
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  • 253
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    Biotechnology and Bioengineering 48 (1995), S. 542-546 
    ISSN: 0006-3592
    Keywords: purification ; inositol monophosphatase ; lithium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol · min-1 · mg-1. The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 ± 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent Km value of the phosphatase for the utilization of inositol-1-phosphate and β-glycerol phosphate are 3.20 × 10-4 and 8 × 10-3 M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K1 was determined to be 6.38 × 10-4 M and the inhibition is uncompetitive. © 1995 John Wiley & Sons, Inc.
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  • 254
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    Biotechnology and Bioengineering 48 (1995), S. 529-541 
    ISSN: 0006-3592
    Keywords: human insulin ; biosynthetic process design ; economic evaluation ; insulin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Human insulin was the first mammalian protein produced in bacteria using recombinant DNA technology. Two technologies were developed; the first based on the separate expression of precursors of chains A and B of insulin, and the second based on the expression of a precursor of proinsulin as a Trp-E fusion protein. Both technologies utilized Escherichia coli as an expression system. Later, a third technology was developed based on a strain of yeast that can secrete a precursor of insulin. The second E. coli process, a variation of which has been commercialized by Eli Lilly and Co., is analyzed in this article from a process design and economic evaluation viewpoint. The objective of this work is to elucidate the technical complexity and high cost associated with the manufacturing of biopharmaceuticals. Human insulin is a good example of a protein-based biopharmaceutical produced in large quantities (a fex tons per year) that requires large scale equipment and presents a multitude of scale-up challenges. Based onthe analysis, a number of conclusions are drawn regarding the cost breakdown and cost dependency on process parameters. Recommendations are made for cost reduction and environmental impact minimization. This analysis was performed using a software tool for computer-aided bioprocess design. © 1995 John Wiley & Sons, Inc.
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  • 255
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    Biotechnology and Bioengineering 48 (1995), S. 551-555 
    ISSN: 0006-3592
    Keywords: enzymatic hydrolysis ; glutamic acid fermentation ; ion-exchange resin column ; product recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Glutamic acid produced from palm waste hydrolysate by fermentation with Brevibacterium lactofermentum ATCC 13869 is produced with a remarkably high yield compared with that produced from pure glucose as a carbon source. The produce yield is 70 g/L with glucose, wherease, when palm waste hydrolysate is the fermentation medium in the same bioreactor under same conditions, it is 88 g/L. The higher yield may be attributed to the fact that this organism has the ability to convert sugars other than only glucose present in the hydrolysate. Bioreactor conditions most conducive for maximum production are pH 7.5, temperature of 30°rmentation period of 48 h, inoculum size 6%, substrate concentration of 10 g per 100 mL, yeast extract 0.5 g per 100 mL as a suitable N source, and biotin at a concentration of 10 pg/L. Palm waste hydrolysate used in this study was prepared by enzymic saccharification of treated palm press fiber under conditions that yielded a maximum of 30 g/L total reducing sugars. Glutamic acid from fermentation broth was recovered by using a chromatographic column (5cm × 60 cm) packed with a strong ion-exchange resin. The filtered broth containing glutamic acid and other inorganic ions was fed to the fully charged column. The broth was continuously recycled at a flow rate of 50 mL/min (retention time of 55 min) until glutamic acid was fully adsorbed on the column leaving other ions in the effluent. Recovery was done by eluting with urea and sodium hydroxide for total displacement of glutamic acid from the resin. The eluent containing 88 g/L of glutamic acid was concentrated by evaporation to obtain solid crystals of the product. © 1995 John Wiley & Sons, Inc.
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  • 256
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    Biotechnology and Bioengineering 48 (1995), S. 592-600 
    ISSN: 0006-3592
    Keywords: Acidianus brierleyi ; pyrite ; bioleaching ; acidophilic thermophile ; metal recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of bioleaching of pyrite (FeS2) by the acidophilic thermophilic bacterium Acidianus brierleyi was studied in a well-mixed batch reactor. Experiments were done at 65°C and pH 1.5 on adsorption of A. brierleyi onto pyrite particles, liquid-phase oxidation of ferrous iron by A. brierleyi, and microbial leaching of pyrite. The adsorption of A. brierleyi was a fast process; equilibrium was attained within the first 30 min of exposure to pyrite. The adsorption equilibrium data were well correlated with the Langmuir isotherm. The oxidation of ferrous iron was markedly accelerated in the presence of A. brierleyi, and the growth yield on ferrous iron was determined. The bioleaching of pyrite by A. brierleyi was found to take place with a direct attack by adsorbed cells on the surface of pyrite, the chemical leaching of pyrite by ferric iron being insignificant. Rate data collected under a wide variety of operating variables were analyzed to determine kinetic and stoichiometric parameters for the microbial pyrite leaching. The specific growth rate on pyrite for A. brierleyi was about four times that for the mesophilic bacterium, Thiobacillus ferrooxidans, whereas the growth yields on pyrite for the two microbes were approximately equal to one another in magnitude. A comparison of A. brierleyi with T. ferrooxidans for pyrite leachability demonstrated the thermophile to be much more effective. © 1995 John Wiley & Sons, Inc.
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  • 257
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    Biotechnology and Bioengineering 48 (1995), S. 614-624 
    ISSN: 0006-3592
    Keywords: biodegradation ; aromatic hydrocarbon ; BTEX ; thermophile ; Thermus ; metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two thermophilic bacteria, Thermus aquaticus ATCC 25104 and Thermus species ATCC 27978, were investigated for their abilities to degrade BTEX (benzene, toluene, ethylbenzene, and xylenes). Thermus aquaticus and the Thermus sp. were grown in a nominal medium at 70°C and 60°C, respectively, and resting cell suspensions were used to study BTEX biodegradation at the same corresponding temperatures. The degradation of BTEX by these cell suspensions was measured in sealed serum bottles against controls that also displayed significant abiotic removals of BTEX under such high-temperature conditions. For T. aquaticus at a suspension density of only 1.3 x 107 cells/mL and an aqueous total BTEX concentration of 2.04 mg/L (0.022 mM), benzene, toluene, ethylbenzene, m-xylene, and an unresolved mixture of o-and p-xylenes were biodegraded by 10, 12, 18, 20, and 20%, respectively, after 45 days of incubation at 70°C. For the Thermus sp. at a suspension density of 1.1 x 107 cells/mL and an aqueous total BTEX concentration of 6.98 mg/L (0.079 mM), benzene, toluene, ethylbenzene, m-xylene, and the unresolved mixture of o-and p-xylenes were biodegraded by 40, 35, 32, 33, and 33%, respectively, after 45 days of incubation at 60°C. Raising the BTEX concentrations lowered the extents of biodegradation. The biodegradations of both benzene and toluene were enhanced when T. aquaticus and the Thermus sp. were pregrown on catechol and o-cresol, respectively, as carbon sources. Use of [U-14C]benzene and [ring-14C]toluene verified that a small fraction of these two compounds was metabolized within 7 days to water-soluble products and CO2 by these nongrowing cell suspensions. Our investigation also revealed that the nominal medium can be simplified by eliminating the yeast extract and using a higher tryptone concentration (0.2%) without affecting the growth and BTEX degrading activities of these cells. © 1995 John Wiley & Sons, Inc.
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  • 258
    ISSN: 0006-3592
    Keywords: interferon ; glycosylation ; CHO cells ; microheterogeneity ; mass spectrometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A recombinant Chinese hamster ovary (CHO) cell line making human interfron-γ (IFN-γ) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-γ was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal2GlcNAc4Man3 which was core ℵl-6 fucosylated at Asn25 but not at Asng97) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal2GlcNAc4Man3 ± Fuc1) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng97 by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. © 1995 John Wiley & Sons, Inc.
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  • 259
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    Biotechnology and Bioengineering 48 (1995), S. 681-698 
    ISSN: 0006-3592
    Keywords: stoichiometry ; biomass yield ; product yield ; metabolic fluxes ; Saccharomyces cerevisiae ; Candida utilis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Using available biochemical information, metabolic networks have been constructed to describe the biochemistry of growth of Saccharomyces cerevisiae and Candida utilis on a wide variety of carbon substrates. All networks contained only two fitted parameters, the P/O ratio and a maintenance coefficient. It is shown that with a growth-associated maintenance coefficient, K, of 1.37 mol ATP/ C-mol protein for both yeasts and P/O ratios of 1.20 and 1.53 for S. cerevisiae and C. utilis, respectively, measured biomass yields could be described accurately. A metabolic flux analysis of aerobic growth of S. cerevisiae on glucose/ethanol mixtures predicted five different metabolic flux regimes upon transition from 100% glucose to 100% ethanol. The metabolic network constructed for growth of S. cerevisiae on glucose was applied to perform a theoretical exercise on the overproduction of amino acids. It is shown that theoretical operational product yield values can be substantially lower than calculated maximum product yields. A practical case of lysine production was analyzed with respect to theoretical bottlenecks limiting product formation. Predictions of network-derived irreversibility limits for Ysp (μ) functions were compared with literature data. The comparisons show that in real systems such irreversibility constraints may be of relevance. It is concluded that analysis of metabolic network stoichiometry is a useful tool to detect metabolic limits and to guide process intensification studies. © 1995 John Wiley & Sons, Inc.
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  • 260
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    Biotechnology and Bioengineering 48 (1995), S. 706-714 
    ISSN: 0006-3592
    Keywords: microencapsulation ; tyrosine production ; tyrosine phenol-lyase ; kinetic data analysis ; ammonia conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The whole cell tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) activity of Erwinia herbicola (ATCC 21434) was microen-capsulated. We studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22°C to 32°C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37°C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42°C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 ± 100 μm, mean ± std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of-the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent KM of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent KM for phenol is 0.5 mM. The apparent KM values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme. © 1995 John Wiley & Sons, Inc.
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  • 261
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    Biotechnology and Bioengineering 45 (1995), S. 18-26 
    ISSN: 0006-3592
    Keywords: hybridoma ; cell death ; chemostat ; autoinhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the present study, the steady-state cell density (X) of chemostat cultures of murine hybridoma was varied by the concentration of glucose and glutamine in culture medium and the dissolved oxygen partial pressure. Except at low glutamine and low oxygen levels, the specific death rate (kd) of the cultures was found to decrease with increasing dilution rate (D). However, the plot of kd vs. X/D yielded linear relation, which suggests that cell death was due to a non-growth-linked inhibitory product of the cells. The kd value measured at low glutamine and low oxygen levels remained practically unchanged over a wide range of D between 0.020 and 0.029 h-1. The kd for low oxygen cultures was always lower than the values obtained in low glucose and low glutamine cultures. A low-molecular-weight component of possibly less than 3000 MW was detected to be cell-death-inducing in the supernatant of exponentially growing cultures. It was neither lactate nor ammonium. The autoinhibitor was not cell-line specific. © 1995 John Wiley & Sons, Inc.
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  • 262
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    Biotechnology and Bioengineering 45 (1995), S. 42-53 
    ISSN: 0006-3592
    Keywords: oxygen uptake rate ; animal cell cultivation ; dissolved oxygen and pH ; state space controller ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k1, respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. © 1995 John Wiley & Sons, Inc.
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  • 263
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    Biotechnology and Bioengineering 45 (1995), S. 27-32 
    ISSN: 0006-3592
    Keywords: esterification ; lipase ; glycerides ; organic solvent ; surfactant ; bioconversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Several surfactant-coated enzymes have been prepared by coating lipases of various origins with a nonionic surfactant, glutamic acid dioleylester ribitol (2C18Δ9GE). Enzymatic interesterification of tripalmitin with oleic acid using the surfactant-coated lipase was carried out in organic media. The surfactant-coated lipases could effectively catalyze the interesterification of glycerides better than did the powder lipases. A suitable organic solvent was an aliphatic hydrocarbon such as isooctane. The enzymatic activity for the interesterification strongly depended on the origin of the lipase. The surfactant-coated lipase prepared by Mucor javanicus showed the highest enzymatic activity for the interesterification of glycerides, although its powder lipase did not show enzymatic activity. Selective interesterification of glycerides could be performed by adjusting the concentration ratio of oleic acid to tripalmitin in isooctane. Di-substituted glyceride could be selectively produced when the concentration ratio of carboxylic acid to glycerides was 7. © 1995 John Wiley & Sons, Inc.
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  • 264
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    Biotechnology and Bioengineering 45 (1995), S. 54-62 
    ISSN: 0006-3592
    Keywords: oxygen uptake rate ; animal cell cultivation ; hybridoma ; monoclonal antibody ; glutamine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Different methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge of KLa, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, a new method of aeration was designed. Oxygen and CO2 transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100-fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O2, CO2, Ar, and N2. The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on-line methods for OUR measurement were tested during the cultivation of animal cells. The new method allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. © 1995 John Wiley & Sons, Inc.
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  • 265
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    Biotechnology and Bioengineering 45 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 266
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    Biotechnology and Bioengineering 45 (1995), S. 86-90 
    ISSN: 0006-3592
    Keywords: hybridoma ; nutrition ; cell death ; apoptosis ; monoclonal antibody ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Association of the availability of nutrients with the phenomenon of programmed cell death - apoptosis - was investigated using hybridoma cells cultured in protein-free medium under conditions of starvation, i.e., in RPMl-1640 medium diluted to 50% with saline. Amino acid mixtures, such as MEM essential amino acids or MEM nonessential amino acids were found to prevent starvation death significantly when added to the diluted medium in 1 to 2 mM concentrations, the MEM vitamin mixture was ineffective, and glutamine displayed a moderate growth-supporting effect. The specific monoclonal antibody production rate in cultures supplemented with amino acid mixtures was strikingly low, whereas supplementation with glutamine alone or simultaneously with other amino acids resulted in a specific antibody production rate comparable with the rate observed in undiluted medium. © 1995 John Wiley & Sons, Inc.
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  • 267
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    Biotechnology and Bioengineering 45 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 268
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    Biotechnology and Bioengineering 45 (1995), S. 165-175 
    ISSN: 0006-3592
    Keywords: pulsed baffled bioreactor ; baffle ; yeast resuspension ; oscillation ; power density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We report experimental data of mass transfer of oxygen into yeast resuspension in a pulsed baffled bioreactor. The bioreactor consists of a 50-mm-diameter column with the presence of a series of either wall (orifice) or central (disc) baffles or a mixture of both where fluid oscillation can also be supermposed during the experiments. Air bubbles are sparged into the bottom of the pulsed baffled bioreactor, and the kinetics of liquid oxygen concentration in the yeast solution is followed using a dissolved oxygen probe with a fast response time of 3 s together with the dynamic gassing-out technique. Among the three different baffle geometries investigated, the orifice baffles gave the highest and sharpest increase in the oxygen transfer rate, and the trends in the kLa measurements are consistent with the fluid mechanics observed within both the systems and previous work. In addition, we have also compared the kLa values with those obtained in a stirred tank; an 11% increase in the KLa is reported. © 1995 John Wiley & Sons, Inc.
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  • 269
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    Biotechnology and Bioengineering 45 (1995), S. 176-186 
    ISSN: 0006-3592
    Keywords: animal cell ; hybridoma cell ; shear ; cell damage ; bioreactor design ; inclination ; bubble column ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Murine hybridoma cells were grown in a bubble column that was inclined up to 45° from vertical. Inclining the column by a few degrees separated the rising bubbles against the upper surface, leaving the bulk of the liquid bubble free. The liquid was circulated well by the rising bubbles, but collection of cells by rising bubbles and exposure of cells to bursting bubbles were minimized. Maximum viable cell count and exponential growth of the cells were not affected by inclination, but an inclination of 30° gave an antibody titer of 42 mg/L, which more than doubled the yield of 17 mg/L in the vertical position. By comparison, the culture gave yields of 30 mg/L when grown in spinner flasks. The enhanced antibody production in the inclined bioreactor corresponded to a prolonged stationary phase of 45 h. © 1995 John Wiley & Sons, Inc.
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  • 270
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    Biotechnology and Bioengineering 45 (1995), S. 187-195 
    ISSN: 0006-3592
    Keywords: interesterification reaction ; surfactant-modified lipase ; modified lipase Saiken ; triglycerides ; fatty acids ; biocatalyst ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. © 1995 John Wiley & Sons, Inc.
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  • 271
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    Biotechnology and Bioengineering 45 (1995), S. 212-218 
    ISSN: 0006-3592
    Keywords: biofilms ; calcium ; anaerobic digestion ; acidogenesis ; lactose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Anerobic biofilms with dominantly acidogenic bacteria were grown in fixed-bed recycle reactors. The influence of calcium concentration in the culture medium on biofilm mass accumulation, immobilized calcium concentration, and biofilm-specific activity was investigated. The results indicate that the biofilm mass accumulation was increased by the presence of calcium in the growth medium when calcium concentration was not higher than 120mg/L. Calcium accumulated in the biofilms increased in proportion to the calcium level in the feed. The biofilms for an increased input calcium concentration showed a trend of decrease in specific activity. The biofilms with a thickeness of less than 0.5 mm had the highest specific activity. The optimum calcium concentration for substrate consumption by the biofilms was 100 to 120 mg/L. The biofilms transferred from higher calcium medium to lower calcium medium were more susceptible to sloughing from their support surfaces, which indicates calcium's role in the stability of the biofilm structure. © 1995 John Wiley & Sons, Inc.
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  • 272
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    Biotechnology and Bioengineering 45 (1995), S. 205-211 
    ISSN: 0006-3592
    Keywords: monoclonal antibodies ; fermentation ; fluidized bed adsorption ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To achive the coarse purification of a monoclonal antibody from whole hybridoma fermentation broth a fluidized bed cation exchange process was used. The procedure consisted of application of the crude sample and washing of the bed in a fluidized mode and elution in a fixed bed mode. A completely clarified eluate was obtained with purification factors between 4 and 8 and a concentration of the desired product (monoclonal antibody) by a factor of more than 3 was achived. Thus, a combination of the three early steps of the downstream process clarification, concentration and coarse purification was possible. Two different materials were tested: a commercially available agarose-based matrix (Stream-line-SP), and a self-derivatized material based on controlled-pore glass (Bioran). Initial experiments were performed to describe the fluidization of the glass material. Comparison with the agarose material showed several differences, the agarose matrix allowing liquid flow closer to plug flow than the glass material. Increased backmixing in the liquid phase was detected when fluidizing the glass adsorbent compared with the agarose-based matrix. Despite this fact, comparison of the two materials with respect to antibody binding and elution demonstrated a similar performance. © 1995 John Wiley & Sons, Inc.
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  • 273
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    Biotechnology and Bioengineering 45 (1995), S. 196-204 
    ISSN: 0006-3592
    Keywords: tryptic casein phosphopeptides ; peptides ; casein phosphopeptides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Anticariogenic casein phosphopeptides (ACPP) contain the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- and have commercial potential as toothpaste, mouthwash, and food additives for the prevention of dental caries. In an approach to develop a commercial-scale process for the production of ACPP we have comprehensively characterized casein phosphopeptides (CPP) produced under industrially relevant conditions. Sodium caseinate (10% w/v) was hydrolyzed by Novo trypsin (commercial grade) at 50°C for 2 h and CPP were purified from the acid clarified hydrolysate by a single-step selective precipitation procedure involving Ca2+ (20 mol/mol casein) and ethanol (50% v/v) at pH 4.6 or 8.0. The individual peptides of the CPP preparations were purified by reversed-phase high-performance liquid chromatography (HPLC) and then identified by amino acid composition and sequence analyses. The yield of the pH 8.0 precipitate (13.85 ± 0.48 wt % of the caseinate) was slightly higher than that of the pH 4.6 precipitate (11.04 ± 0.30 wt % of the caseinate). However, the pH 4.6 precipitate contained predominantly (86.4 mol %) ACPP cluster peptides with small amounts of the diphosphorylated peptides (13.6 mol %), αs1(43-58) and αs2(126-136). In the pH 8.0 precipitate the cluster peptides represented a smaller proportion of the total peptides (61.9 mol %) due to increased recoveries of the diphosphorylated peptides (24.4 mol %) as well as the additional recovery of the monophosphorylated peptide β(33-48) (13.7 mol %) indicating increased cross-linking by Ca2+ at the higher pH. The recovery of the ACPP from the original caseinate was similar for both the pH 4.6 and 8.0 precipitates. Slight chymotryptic activity was detected in the industrial-grade enzyme, resulting in minor truncation of some peptides. Also some deamidation and methionine oxidation of one peptide, αs1(59-79), were detected. In conclusion, ACPP can be produced under industrially relevant conditions with only minor modifications such as slight truncation, deamidation, and methionine oxidation. However, in order to prepare casein phosphopeptides predominantly containing the cluster sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, the single-step selective precipitation with Ca2+/ethanol should be performed at pH 4.6 rather than pH 8.0. © 1995 John Wiley & Sons, Inc.
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  • 274
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    Biotechnology and Bioengineering 45 (1995), S. 245-250 
    ISSN: 0006-3592
    Keywords: Escherichia coli enterotoxin ; fed batch ; high cell density ; fermentation ; purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: High cell density fermentation studies were performed to produce the B subunit of Escherichia coli heat-labile enterotoxin (LTB) from a Vibrio cholerae culture that carries a recombinant plasmid with an ampicillin resistance gene, tac promoter, and the gene encoding LTB. Upon induction with isopropyl-β-D-thiogalactopyranoside (IPTG) the culture secreted the protein into the extracellular milieu. Fed-batch fermentation with stepwise addition of a total of 5 mM of IPTG during the active growth phase of the organism resulted in the production of 400 mg/L of LTB in 9 h and a cell optical density (OD) of 24. The LTB was purified to homogeneity with 70% recovery from the fermentation broth and was found to be chemically and biologically identical to the native protein by N-terminal amino acid sequencing and receptor binding assay. © 1995 John Wiley & Sons, Inc.
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  • 275
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    Biotechnology and Bioengineering 45 (1995), S. 239-244 
    ISSN: 0006-3592
    Keywords: cellulase ; newsprint ; deinking sludge ; surfactant ; hydrolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Disposal of sludge from deinking mills represents a significant proportion of operating costs. Bioconversion of the cellulosic fraction of deinking sludge (DIS) to ethanol greatly reduces disposal costs while producing an environmentally friendly fuel. In this study, the cellulosic fraction of newsprint and deinking sludge was hydrolysed to produce fermentable sugars. For newsprint, a particle size of 1 to 1.5 mm provided optimal reaction rates in batch reactors over practical hydrolysis times, and reducing sugar concentrations as high as 35 g/L could be achieved using a fed-batch reactor configuration. For both newsprint and DIS, the hydrolysis rate increased nonlinearly with enzyme loading. Tween-80 only marginally improved sugar production but was able to release sugars from cellulosic substrates in the absence of lytic enzymes, in an amount proportional to the surfactant concentration and the substrate particle size. DIS was relatively recalcitrant to enzymatic hydrolysis, possibly due in part to inhibition by hydrophobic constituents. © 1995 John Wiley & Sons, Inc.
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  • 276
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    Biotechnology and Bioengineering 45 (1995), S. 219-228 
    ISSN: 0006-3592
    Keywords: formate conversion ; mass spectrometer ; anaerobic conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The dynamics of the anaerobic conversion of formate in a microbial mixed culture taken from an anaerobic fluidized bed reactor was studied using a new stirred micro reactor equipped with a membrane mass spectrometer. The microreactor with a toroidally shaped bottom and pitched blade turbine and a cylindrical flow guide was thermostated and additionally equipped with a pH electrode and pH control. During fed-batch experiments using formate, the dissolved gases (methane, hydrogen, and carbon dioxide), as well as the acid consumption rates for pH control were monitored continuously. Initially and at the end of each experiment, organic acids were analyzed using ion chromatography (IC). It was found that about 50% of the formate was converted to methane via hydrogen and carbon dioxide, 40% gave methane either directly or via acetate. This was calculated from experiments using H13CO3- pulses and measurement of 12CH4 and 13CH4 production rates. About 10% of the formate was converted to lactate, acetate, and propionate, thereby increasing the measured CO2/CH4 production ratio. The nondissociated formic acid was shown to be rate determining. From the relatively high Ks value of 2.5 mmol m-3, it was concluded that formate cannot play an important role in electron transfer. During dynamic feeding of formate, hydrogen concentration always increased to a maximum before decreasing again. This peak was found to be very discriminative during modeling. From the various models set up, only those with two-stage degradation and double Monod kinetics, both for CO2 and hydrogen, were able to describe the experimental data adequately. Additional discrimination was possible with the IC measurement of organic acids. © 1995 John Wiley & Sons, Inc.
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  • 277
    ISSN: 0006-3592
    Keywords: methanotroph ; methane monooxygenase ; nitrogenase ; hydrogenase ; batch culture conditions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch culture conditions were established for the formation of H2-driven whole-cell soluble or particulate methane monooxygenase (sMMO or pMMO) activity in the obligate methanotroph, Methylosinus trichosporum Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H2 to a nitrate-containing minimal salts growth medium or Ni and Mo to a nitrate-lacking growth medium (induces a nitrogenase that generates intracellular H2) markedly enhanced both the hydrogenase and the accompanying washed-cell H2-driven MMO activities of shake-flask cultured cells. For sMMO containing cells, H2 provided in vitro reducing power for the oxidation of chlorinated solvents such as chloroform and trichloroethylene. Cell cultivations under N2-fixing conditions in a 5-L bioreactor, however, required an initial nitrate concentration of at least 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H2-driven sMMO or pMMO activity. Elevation of the initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalent growth yield. High nitrate also shortened the culture time for sMMO containing cells by ∼25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting-state cells retained 90% and 70% of their H2-driven sMMO and pMMO activity, respectively. This makes their practical use quite feasible. © 1995 John Wiley & Sons, Inc.
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  • 278
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    Biotechnology and Bioengineering 45 (1995), S. 251-260 
    ISSN: 0006-3592
    Keywords: macroalgal cells ; stirred-tank bioreactor ; photolithotrophic cultivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Filamentous cell cultures derived from female gametophytes of the temperate brown macroalga Laminaria saccharina were photolithotrophically cultivated in artificial seawater medium within an illuminated 1.3-L stirred-tank bioreactor at 13°C using CO2 in air as the carbon source. A Monod model adequately described light-saturated growth. The apparent half-saturation constant (Ko) was 23 μE/m2-s, and maximum specific growth rate was 0.15 day-1. At a constant inoculation cell density of 50 mg DCW/L, biomass productivity after 26 days of cultivation increased from 630 mg DCW/L at 18 μE/m2-s to 890 mg DCW/L at 228 μE/m2-s. At 98 μE/m2-s, 1.1 vvm aeration rate, and 250 rpm impeller speed, the CO2 transfer rates (CO2 TRs) and CO2 consumption rates (rco2) were determined over the cultivation period. At peak CO2 demand, the maximum CO2 TR was 0.19 mmol CO2/L-h, but rco2 was only 0.15 mmol CO2/L-h, implying that the culture was not CO2 transport limited. This is the first reported bioreactor cultivation study of cell cultures derived from a macrophytic marine alga. © 1995 John Wiley & Sons, Inc.
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  • 279
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    Biotechnology and Bioengineering 45 (1995), S. 261-267 
    ISSN: 0006-3592
    Keywords: Biotransformations in aqueous suspensions ; chymotrypsin ; penicillin amidase ; immobilized enzyme ; peptide synthesis ; D-phenylglycine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH2 with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by α-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be 〉90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. © 1995 John Wiley & Sons, Inc.
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  • 280
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    Biotechnology and Bioengineering 45 (1995), S. 276-278 
    ISSN: 0006-3592
    Keywords: enzymic peptide synthesis ; solvent free system, chymotrypsin ; thermolysin ; peptides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Thermolysin-catalyzed (EC 3.4.24.4) and chymotrypsin-catalyzed (EC 3.4.21.1) peptide synthesis reactions were accomplished without any organic solvent in the presence of low amounts of water under sonication and fluidization. The systems used are considered to be microaqueous solvent-free ones. The influence of several reaction parameters, such as time, the amount of enzyme, the amount of water in free form or bound as hydration water, and the N/C component ratio, on the vield of the thermolysin-catalyzed synthesis of Z-Phe-Leu-NH2 (up to 87% yield) was investigated in a sonicated system. Besides Z-Phe-Leu-NH2, the tripeptide derivatives Ac-Xaa-Trp-Leu-NH2, (Xaa = Gly, Ala) were also obtained in good yields of 79 and 71% respectively. In the latter case, no hydrolytic side reactions were observed. Using a fluidized-bed reactor, chymotrypsin- and thermolysin-catalyzed syntheses of N-protected di- and tripeptide amides could be perfromed with yields in the range of 10 to 40%. © 1995 John Wiley & Sons, Inc.
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  • 281
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    Biotechnology and Bioengineering 45 (1995), S. 268-275 
    ISSN: 0006-3592
    Keywords: poly(D-3-hydroxybutyrate) ; P(3HB) ; Alcaligenes eutrophus ; gas explosion ; autotroph ; hydrogen oxidizing bacterium ; carbon dioxide fixation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Hydrogen-oxidizing bacterium, Alcaligenes eutrophus autotrophically produces biodegradable plastic material, poly(D-3-hydroxybutyrate), P(3HB), from carbon dioxide, hydrogen, and oxygen. In autotrophic cultivation of the microorganism, it is essential to eliminate possible occurrence of gas explosions from the fermentation process. We developed a bench-plant scale, recycled-gas, closed-circuit culture system equipped with several safety features to perform autotrophic cultivation of A. eutrophus by maintaining the oxygen concentration in the substrate gas phase below the lower limit for a gas explosion (6.9%). The culture vessel utilized a baskettype agitator, resulting in a KL a value of 2970 h-1. Oxygen gas was also directly fed to the fermentor separately from the other gases. As a result, 91.3 g · dm-3 of the cells and 61.9 g · dm-3 of P(3HB) were obtained after 40 h of cultivation under this oxygen-limited condition. The results compared favorably with those reported for mass production of P(3HB) by heterotrophic fermentation. © 1995 John Wiley & Sons, Inc.
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  • 282
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    Biotechnology and Bioengineering 45 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 283
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    Biotechnology and Bioengineering 45 (1995), S. 279-284 
    ISSN: 0006-3592
    Keywords: carbon tetrachloride ; nitrate inhibition ; biodegradation ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of nitrate inhibition of carbon tetrachloride (CT) transformation were examined using a denitrifying consortium. Comparison of data from fed-batch experiments to the model reported by Hooker et al. indicate that the inhibition constant ranges between 3.2 and 21 mg/L, with an average of 8.8 mg/L. This range is much lower than the previously reported value of 169 mg/L. Simulations using the corrected parameter accurately reflect this new data and the data reported by Hooker et al. In contrast, the earlier reported coefficient value does not reflect the data reported in this work. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 45 (1995), S. 285-291 
    ISSN: 0006-3592
    Keywords: Klebsiella pneumoniae 62-1 ; isochorismate hydroxymutase (E.C. 5.4.99.6) ; affinity immobilization ; isochorismate excretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two methods are described for the preparation of enantiomerically pure (+)-trans-isochorismic acid, an important metabolite of the postchorismate pathway. Both methods can be employed to prepare isotopically labeled isochorismic acid. One of the two methods is suitable to prepare bulk quantities of isochorismic acid using a recombinant strain of Klebsiella pneumoniae 62-1. © 1995 John Wiley & Sons, Inc.
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  • 285
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    Biotechnology and Bioengineering 45 (1995), S. 292-303 
    ISSN: 0006-3592
    Keywords: fluxes ; intracellular fluxes ; hybridoma cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Intracellular fluxes are important in defining cellular physiology and its changes in response to environmental variations. Stoichiometric balances combined with extra cellular metabolite measurements were applied to the estimation of intracellular fluxes and the study of energy metabolism in the hybridoma cell line ATCC CRL 1606. Redundant measurements allowed the evaluation of the consistency of the stoichiometry, measurements, and pseudo-steady-state assumption leading to refinement of the assumed biochemistry and identification of measurement errors. To validate the flux estimates, two batch experiments were performed with glucose labeled in the 1 position with 13C. The distribution of 13C in secreted lactate was measured via nuclear magnetic resonance spectroscopy (NMR) and compared to that predicted from the estimated intracellular fluxes. There was good agreement between the measured and estimated isotope distributions, demonstrating the validity of the flux estimates obtained from stoichiometric balances. © 1995 John Wiley & Sons, Inc.
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  • 286
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    Biotechnology and Bioengineering 45 (1995), S. 304-309 
    ISSN: 0006-3592
    Keywords: phenol ; substituted phenol ; tyrosinase ; immobilization ; chitosan ; coagulant ; immobilized enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of phenols and aromatic amines from industrial wastewater by tyrosinase was investigated. A color change from colorless to darkbrown was observed, but no precipitate was formed. Colored products were found to be easily removed by a combination treatment with tyrosinase and a cationic polymer coagulant containing amino group, such as hexamethylenediamine-epichlorohidrin polycondensate, polyethleneimine, or chitosan. The first two coagulants, synthetic polymers, were more effective than chitosan, a polymer produced in crustacean shells. Phenols and aromatic amines are not precipitated by any kind of coagulants, but their enzymatic reaction products are easily precipitated by a cationic polymer coagulant. These results indicate that the combination of tyrosinase and a cationic polymer coagulant is effective in removing carcinogenic phenols and aromatic amines from an aqueous solution. Immobilization of tyrosinase on magnetite gave a good retention of activity (80%) and storage stability i.e., only 5% loss after 15 days of storage at ambient temperature. In the treatment of immobilized tyrosinase, colored enzymatic reaction products were removed by less coagulant compared with soluble tyrosinase. © 1995 John Wiley & Sons, Inc.
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  • 287
    ISSN: 0006-3592
    Keywords: chimeric antibodies ; transfectoma cells ; hollow fiber fermentor ; immunoglobulin enhancer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt)and the neomycin (neo) selection marker genes. ELISA-based screening of transfectoma clones resulted in the isolation of IgG-producing transfectomas. Introduction of the kappa light-chain 3′-enhancer into the light-chain expression vector significantly increased immunoglobulin expression, but only when the enhancer was located at its physiological site, 9 kb downstream of the kappa constant region exon. With some of the transfectomas, final yields of up to 80 mg/L of chimeric IgG were obtained in conventional flask cultures using serum-free growth medium. A pilot-scale AcuSyst Maximizer hollow fiber cell culture system was used for the production of gram amounts of chimeric IgG. Results obtained with different transfectoma clones in conventional culture were not fully predictive for yields in the hollow fiber system. In contrast, differences in productivity between individual clones in the laboratory-scale Tecnomouse cell culture unit were comparable with those in the Maximizer system. Up to 200 mg of chimeric IgG were produced per day in one Maximizer bioreactor. © 1995 John Wiley & Sons, Inc.
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  • 288
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    Biotechnology and Bioengineering 45 (1995), S. 328-336 
    ISSN: 0006-3592
    Keywords: cellulase recycling ; lignin ; lignocellulose hydrolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recycling of cellulases should lower the overall cost of lignocellulosiic bioconversion processes. In this study, three recycling strategies were evaluated to determine their efficiencies over five successive rounds of hydrolysis. The effect of lignin on recycling was examined by comparing water-washed, steam-exploded birch (WB; 32% lignin) and WB which had been further extracted with alkali and peroxide (PB; 4% lignin). When the cellulases were recovered from the residual substrates after partial hydrolysis of both substrates, the recovered cellulase activity toward the mixture of fresh and residual substrates decreased after each recycling step. When the cellulases in the supernatants were also recycled, up to 20% more activity could be recovered. In both of these cases, the recovered activities did not correspond to the activities expected from the amount of cellulase protein recovered during recycling. The best recovery was obtained when the cellulases were recovered from both the residue and the supernatant after complete hydrolysis of the PB substrate. In this case, all of the originally added cellulase activity could be recovered for four consecutive hydrolysis rounds. However, when the same recycling strategy was carried out using the WB substrate, the recovered cellulase activity declined quickly with each recycling round. In all three of the recycling strategies, lower cellulase activities were recovered from the substrates with higher lignin contents. © 1995 John Wiley & Sons, Inc.
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  • 289
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    Biotechnology and Bioengineering 45 (1995), S. 337-343 
    ISSN: 0006-3592
    Keywords: dielectrophoresis ; cells, separation of ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Dielectrophoresis is the movement of particles in non-uniform alternating and direct current (AC, DC) electric fields. When nonuniform electric fields are created between microelectrodes, cells will redistribute themselves around the electrodes, the force holding the cells in place dependig on the local electric field and on the electrical properties of the cells themselves and the suspending medium. Steric drag forces produced by a gentle fluid flow in the chamber can be used to separate cells by selectively lifting cells from potential energy wells produced by the electric field. The technique is demonstrated in the batch separation of bacteria, yeast cells, and plant cells. Continuous separation and extraction of two cell types can be achieved by repeated reversing of the fluid flow direction in phase with the switching on and off of the applied voltage, and the efficacy of the technique is demonstrated for viable and nonviable (heat-treated) yeast cells. © 1995 John Wiley & Sons, Inc.
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  • 290
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    Biotechnology and Bioengineering 45 (1995), S. 320-327 
    ISSN: 0006-3592
    Keywords: cell recycle reactor ; ultrafiltration tubular membranes ; high lactic acid productivities ; best operational conditions ; different dilution rates ; start-up strategy ; membrane permeability ; long-term fermentations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h-1, D = 0.40 h-1, or D = 0.58 h-1). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h-1 being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at “steady state,” the membrane permeability being kept around 15 L/m2 h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. © 1995 John Wiley & Sons, Inc.
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  • 291
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    Biotechnology and Bioengineering 45 (1995), S. 344-355 
    ISSN: 0006-3592
    Keywords: esterification ; Chromobacterium viscosum ; lipase ; microemulsions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chromobacterium viscosum (CV) lipase solubilized in water-in-oil (w/o) microemulsions based on the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been used for multigram-scale ester synthesis, including the kinetic resolution of a secondary alcohol. The stability of CV lipase in all the CTAB microemulsions studied was excellent and was superior to that observed in aqueous buffer at the same pH and temperature. Kinetic studies were performed using the synthesis of ethylhexadecanoate as a model reaction. Under pseudo-first-order conditions, the synthesis rates were linearlydependent on the enzyme and fatty acid concentrations and the R dependence shows the characteristic bell-shaped curve (where R = [H2O]/[surfactant]). The dependence of enzyme activity toward octyldecanoate synthesis on the pH of the dispersed buffer phase is in marked contrast to that observed for the pH dependence of CV lipase toward p-nitrophenylbutyrate hydrolysis. In the former case, the pH-activity profile is approximately sigmoidal, which may reflect the ionization state of the fatty acid substrate. In the latter case, the pH dependence is minimal at both R = 10 and R = 50, suggesting the enzyme does not experience a changed pH environment. Inclusion of a pH-sensitive probe molecule into those incubations containing fatty acid clearly demonstrates that the probe molecule experiences a changed environment consistent with that expected for the selected buffer. An in situ Fourier transform nuclear magnetic resonance (FT-NMR) assay has been developed which allows continuous monitoring of the esterification reactions, thereby providing an additional means of determining initial rates. The method may be of general value for lipase assays in microemulsions since it may provide, at the same time, information regarding enzyme regioselectivity. © 1995 John Wiley & Sons, Inc.
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  • 292
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    Biotechnology and Bioengineering 45 (1995), S. 356-365 
    ISSN: 0006-3592
    Keywords: Escherichia coli KO11 ; ethanol production ; kinetic model ; lignocellulosic hydrolysate ; fermentation, mixed sugar ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fermentation kinetics for separate as well as simultaneous glucose and xylose fermentation with recombinant ethanologenic Escherichia coli KO11 are presented. Glucose and xylose were consumed simultaneously and exhibited mutual inhibition. The glucose exhibited 15 times stronger inhibition in xyclose fermentation than vice versa. The fermentation of condensate from steampretreated willow (Salix) was investigated. The kinetics were studied in detoxified as well as in nondetoxified condensate. The fermentation of the condensate followed two phases: First the glucose and some of the pentoses (xylose in addition to small amounts of arabinose) were fermented simultaneously, and then the remaining part of the pentoses were fermented. The rate of the first phase was independent of the detoxification method used, whereas the rate of the second phase was found to be strongly dependent. When the condensate was detoxified with overliming in combination with sulfite, which was the best detoxification method investigated, the sugars in the condensate, 9 g/L, were fermented in 11 h. The same fermentation took 150 h in nondetoxified condensate. The experimental data were used to develop an empirical model, describing the batch fermentation of recombinant E. coli KO11 in the condensate. The model is based on Monod kinetics including substrate and product inhibition and the sum of the inhibition exerted by the rest of the inhibitors, lumped together. © 1995 John Wiley & Sons, Inc.
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  • 293
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    Biotechnology and Bioengineering 45 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 294
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    Biotechnology and Bioengineering 45 (1995), S. 366-373 
    ISSN: 0006-3592
    Keywords: enzymatic hydrolysis ; cellulase ; polyoxyalkylene ; adsorption ; reactive two-phase partition ; solubilization in organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cellulase was modified with amphilic copolymers made of α-allyl-ω-methoxy polyoxyalkylene (POA) and maleic acid anhydride (MAA) to improve the cellulose hydrolytic reactivity and cellulase separation. Amino groups of the cellulase molecule are covalently coupled with the MAA functional groups of the copolymer. At the maximum degree of modification (DM) of 55%, the modified cellulase activity retained more than 80% of the unmodified native cellulase activity. The modified cellulase shows greater stability against temperature, pH, and organic solvents, and demonstrated greater conversion of substrate than native cellulase does. Cellulase modification is also useful for controlling strong adsorption of cellulase onto substrate. Moreover, cellulase modified with the amphiphilic copolymer displays different separation characteristics which are new. One is a reactive two-phase partition and another is solubility in organic solvents. It appears that these characteristics of modified cellulase work very effectively in the hydrolysis of cellulose as a total system, which constitutes the purification of cellulase from culture broth, hydrolysis of cellulose, and recovery of cellulase from the reaction mixture. © 1995 John Wiley & Sons, Inc.
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  • 295
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    Biotechnology and Bioengineering 45 (1995), S. 374-378 
    ISSN: 0006-3592
    Keywords: cell culture ; patterened surfaces ; cell adhesion ; hydrogel ; polyHEMA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chinese hamster ovary (CHO) cells were attached to tiny adhesive sites in poly-2-hydroxyethyl methacrylate(polyHEMA-) coated glass, and their divison properties were examined. The adhesive sites were produced by placing a metal mask, containing 8-μm-diameter holes arranged in a regular pattern, on top of the coated glass and exposing the sandwich to glow discharge treatment. This treatment produced an ordered array of circular cavities in the polyHEMA down to the glass. These adhesive sites were smaller in diameter than a newborn CHO cell, so that, upon division, there would theoretically be room for only one of the two new daughter cells to remain attached. It was found that individual CHO cells attached to, and grew upon, the sites, and that division normally resulted in the releas of one of the two new daughters. It is concluded that this culture technique has applications in research on the mammalian cell cycle, cell partitioning, and cellular senescence. © 1995 John Wiley & Sons, Inc.
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  • 296
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    Biotechnology and Bioengineering 45 (1995), S. 387-397 
    ISSN: 0006-3592
    Keywords: transesterification ; water activity ; lipolytic enzymes ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (aw). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters. © 1995 John Wiley & Sons, Inc.
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  • 297
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    Biotechnology and Bioengineering 45 (1995), S. 398-405 
    ISSN: 0006-3592
    Keywords: anaerobic digestion ; full-scale ; granule activity ; multiplate reactor ; solid retention ; whey permeate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A 450-m3 multiplate anaerobic reactor (MPAR) has been started-up in April 1992 for treating wastewater (whey permeate and domestic wastewater) at the Nutrinor (Lactel) cheese factory in Chambord (Québec, Canada). The MPAR consists of four superimposed sections. The liquid flows upwards from one section to the next, while the gas is collected below each plate and evacuated through side-outlets. The wastewater is concurrently distributed at the bottom of the first, second, and third sections, as 50%, 33%, and 17% of the total influent stream, respectively. Granular anaerobic sludge at an initial concentration of 30 kg of volatile suspended solids (VSS) per cubic meter of reactor liquid volume was used to inoculate the reactor. Under normal operation of the factory, the chemical oxygen demand (COD) concentration of the influent ranged from 20 to 37 kg COD m-3. The reactor organic loading rate (OLR) fluctuated between 9 and 14.7 kg COD m-3 d-1 for hydraulic retention times (HRT) maintained between 55 and 68 h. At the highest OLR, the MPAR showed an efficiency of 98% and 92% for soluble and total COD removal, respectively, and a methane production rate averaging around 4 m3 m-3 d-1.Biomass-specific activities ranged between 7 and 51, 1.3 and 8.5, 5.3 and 12.2, 60 and 119, and 119 and 211 mmol g-1 VSS d-1 for glucose, propionate, acetate, formate, and hydrogen, respectively. Average equivalent-diameter of the granules was around 0.65 mm. The MPAR reactor generally showed a large capacity for solid retention with a biomass content between 32 and 37 kg VSS m-3. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 45 (1995), S. 379-386 
    ISSN: 0006-3592
    Keywords: propionic acid fermentation ; Propionibacterium acidipropionici ; immobilized cell bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took ∼55 h to ferment whey permeate containing ∼45 g/L lactose to ∼20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L · h to 0.47 g/L · h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L · g cell. The productivity increased to 0.68 g/L · h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 45 (1995), S. 415-425 
    ISSN: 0006-3592
    Keywords: plant cell suspension culture ; capillary shear loop ; Morinda citrifolia ; shear susceptibility ; morphology ; stirred tank reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The shear susceptibility of cell suspension cultures of the plant cell Morinda citrifolia was investigated by subjecting the cells to the well-defined shear field generated in turbulent flow through a capillary. Suspensions were circulated using a peristaltic pump and average shear stresses between 25 and 350 N m-2 were generated in the capillary test section. Control experiments were performed to assess the possible contribution of the peristaltic pump to the observed cell damage. There was clear evidence of pump-induced damage at the more severe test conditions and all viability measurements were corrected accordingly. Both shake flask suspension cultures (aged between 9 and 15 days) and repeated batch fermentation cultures, grown in a stirred tank reactor (STR) under a variety of controlled agitation conditions, were tested in the capillary shear loop. The cell damage incurred was evaluated in terms of suspension viability, as determined by a dye exclusion technique. Viability loss was found to conform closely to a first-order model in which the rate constant was observed to increase with the imposed shear stress. Furthermore, a linear relationship was identified between the specific death constant and the cumulative energy dissipated. Post-shear morphological measurements showed that the chain length distribution is shifted toward markedly lower values. In comparison with shake flask cultures, repeated batch fermentation cultures exhibited a marked increase in sensitivity to capillary shear. Based upon the determined morphological characteristics, this result is primarily attributable to the increased chain lengths characteristic of the repeated batch cultures. © 1995 John Wiley & Sons, Inc.
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  • 300
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    Biotechnology and Bioengineering 45 (1995), S. 406-414 
    ISSN: 0006-3592
    Keywords: chymotrypsin ; differential scanning calorimetry ; ligands ; lipase ; organic media ; sorbitol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (aw = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89°C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72°C). © 1995 John Wiley & Sons, Inc.
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