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  • Electronic Resource  (3,075)
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  • 1990-1994  (3,075)
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  • 101
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 102
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 412-425 
    ISSN: 0887-3585
    Keywords: normal modes ; constraint dynamics ; molecular dynamics ; lysozyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Analysis of extended molecular dynamics (MD) simulations of lysozyme in vacuo and in aqueous solution reveals that it is possible to separate the configurational space into two subspaces: (1) an “essential” subspace containing only a few degrees of freedom in which anharmonic motion occurs that comprises most of the positional fluctuations; and (2) the remaining space in which the motion has a narrow Gaussian distribution and which can be considered as “physically constrained.” If overall translation and rotation are eliminated, the two spaces can be constructed by a simple linear transformation in Cartesian coordinate space, which remains valid over several hundred picoseconds. The transformation follows from the covariance matrix of the positional deviations. The essential degrees of freedom seem to describe motions which are relevant for the function of the protein, while the physically constrained subspace merely describes irrelevant local fluctuations. The near-constraint behavior of the latter subspace allows the separation of equations of motion and promises the possibility of investigating independently the essential space and performing dynamic simulations only in this reduced space. © 1993 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 103
    ISSN: 0887-3585
    Keywords: transcription factors ; zinc finger ; generalized order parameter ; effective correlation time ; internal protein motions ; Lipari-Szabo model ; “model-free” approach ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on 15N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S2) and an effective correlation time (τe) for the internal motion of each amide bond. A back calculation, using the same model, yielded the {1H}-15N nuclear Overhauser effects (NOEs) and the 15N spin-lattice relaxation times (T1) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major α-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S2-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental τe-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (〈0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower τe-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487. © 1993 Wiley-Liss, Inc.
    Additional Material: 12 Ill.
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  • 104
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 426-434 
    ISSN: 0887-3585
    Keywords: enolase ; mutagenesis ; activity ; enzyme ; active site ; charge shuttle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Yeast has two enolase isozymes (called 1 and 2), either of which suffices for growth. We cloned DNA encoding the enolase 1 protein coding and promoter regions flanked by BamHI termini using the PCR. The DNA, which contained no nucleotide base changes altering the protein sequence, was cloned into the multicopy shuttle vector pRS314 and transformed into a yeast strain with a deletion in its enolase 1 gene. The resulting plasmid-containing strain makes enolase 1 in quantities which depend on cell growth. A ‘charge shuttle’ mechanism of action of enolase based on X-ray crystallographic evidence (Lebioda and Stec, Biochemistry 30 : 2817, 1991) involves Glu-168 accepting a proton from a water molecule that in turn accepts a proton from a carbon-2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide-directed site-directed mutagenesis. Its identity was confirmed by N-terminal sequence analysis, HPLC on Superose 12, SDS-gel electrophoresis, and the sequence of the mutated DNA protein-coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds substrate/product, AEP (3-aminoenolpyruvate-2-phosphate, the 3-amino analogue of the product phosphoenolpyruvate) and TSP (D-tartronate semialdehyde-2-phosphate, the aldehyde analogue of the substrate 2-phosphoglycerate), the latter two at least with affinities similar to those of the native enzyme. The E168Q enolase also produces absorbance changes in the analogues. The reaction with AEP is consistent with the ‘charge shuttle’ mechanism; the reaction with TSP, which presumably requires proton removal from carbon-2, is complex but shows a very slow phase consistent with expectations. © 1993 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 105
    ISSN: 0887-3585
    Keywords: metalloproteinase ; inhibitor ; X-ray diffraction ; tumor invasion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A nonglycosylated (N30QN78Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P21, with cell dimensions a = 35.28, b = 53.95, c = 48.56, and β = 96.0°. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 Å resolution. Complete data have been collected to 2.9 Å and a search for heavymetal derivatives is in progress. © 1993 Wiley-Liss, Inc.
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  • 106
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 107
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 108
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 360-373 
    ISSN: 0887-3585
    Keywords: conformational substates ; protein conformations ; molecular dynamics ; nonbanded relaxations ; ergodic measures ; s-peptide ; RNase A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The dynamic properties of the RNase A/3′-UMP enzyme/product complex and the S-peptide of RNase A have been investigated by molecular dynamics simulations using suitable generalization of ideas introduced to probe the energy landscape in structural glasses. We introduce two measures, namely, the kinetic energy fluctuation metric and the force metric, both of which are used to calculate the time needed for sampling the conformation space of the molecules. The calculation of the fluctuation metric requires a single trajectory whereas the force metric is computed using two independent trajectories. The vacuum MD simulations show that for both systems the time required for kinetic energy equipartitioning is surprisingly long even at high temperatures. We show that the force metric is a powerful means of probing the nature and relative importance of conformational substates which determine the dynamics at low temperatures. In particular the time dependence of the non-bonded force metric is used to demonstrate that at low temperatures the system is predominantly localized hi a single cluster of conformational substates. The force metric is used to show that relaxation of long range (in sequence space) interactions must be mediated by a sequence of local dihedral angle transitions. We also argue that the time needed for compact structure formation is intimately related to the time needed for the relaxation of the dihedral angle degrees of freedom. The tame for non-bonded interactions, which drive protein molecules to fold under appropriate conditions, to relax becomes extremely long as the temperature is lowered suggesting that the formation of maximally compact structure hi proteins must be a very slow process. © 1993 Wiley-Liss, Inc.
    Additional Material: 15 Ill.
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  • 109
    ISSN: 0887-3585
    Keywords: crambin ; terative relaxation matrix approach ; ensemble averaging ; solution structure ; restrained molecular dynamics ; stereospecific assignments ; 2D NMR ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure in solution of crambin, a small protein of 46 residues, has been determined from 2D NMR data using an iterative relaxation matrix approach (IRMA) together with distance geometry, distance bound driven dynamics, molecular dynamics, and energy minimization. A new protocol based on an “ensemble” approach is proposed and compared to the more standard initial rate analysis approach and a “single structure” relaxation matrix approach. The effects of fast local motions are included and R-factor calculations are performed on NOE build-ups to describe the quality of agreement between theory and experiment. A new method for stereospecific assignment of prochiral groups, based on a comparison of theoretical and experimental NOE intensities, has been applied. The solution structure of crambin could be determined with a precision (rmsd from the average structure) of 0.7 Å on backbone atoms and 1.1 Å on all heavy atoms and is largely similar to the crystal structure with a small difference observed in the position of the side chain of Tyr-29 which is determined in solution by both J-coupling and NOE data. Regions of higher structural variability (suggesting higher mobility) are found hi the solution structure, in particular for the loop between the two helices (Gly-20 to Pro-22). © 1993 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 110
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 138-151 
    ISSN: 0887-3585
    Keywords: structure prediction ; computer modeling ; structure comparison ; sequence identity ; protein homology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledgebased approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict. © 1993 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 111
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 17 (1993), S. 152-160 
    ISSN: 0887-3585
    Keywords: rubredoxin ; redox potential ; ironsulfur protein ; protein relaxation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A theoretical investigation of the protein contribution to the redox potential of the iron-sulfur protein rubredoxin is presented. Structures of the oxidized and reduced forms of the protein were obtained by energy minimizing the oxidized crystal structure of Clostridium pasteurianum rubredoxin with appropriate charges and parameters. By including 102 crystal waters, structures close to the original crystal structure were obtained (rms difference of 1.16 Å), even with extensive minimization, thus allowing accurate calculations of comparative energies. Our calculations indicate an energy change of about -60 kcal/mol (2.58 eV) in the protein alone upon reduction. This energy change was due to both the change in charge of the redox site and the subsequent relaxation of the protein. An energy minimization procedure for the relaxation gives rms differences between the oxidized and reduced states of about 0.2 Å. The changes were small and occurred in both the backbone and sidechain mainly near the Fe-S center but contributed about - 16 kcal/mol (0.69 eV) to the total protein contribution. Although the neglect of certain effects such as electronic polarization may make the relaxation energies calculated an upper limit, the results indicate that protein relaxation contributes substantially to the redox potential. © 1993 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
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  • 112
    ISSN: 0887-3585
    Keywords: refined structures ; mutant ; substrate binding ; zinc coordination ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A complex of carbonic anhydrase (CA) with one of its substrates, bicarbonate, has been studied crystallographically. Human isoenzyme II was mutated at position 200 from threonine to histidine, which results in higher affinity for bicarbonate. The HCO3- ion binds in the active site to the zinc ion as a pseudo-bidentate ligand which gives the metal a coordination geometry between tetrahedral and trigonal bipyramide. The water/hydroxide normally bound with tetrahedral coordination to the zinc is probably replaced by the OH group of the bicarbonate ion. The importance of residues Thr-199 and Glu-106 in controlling the binding orientation of HCO3- is discussed as well as the catalytic mechanism. Both the complex as well as the uncomplexed mutant were studied at 1.9 Å resolution. © 1993 Wiley-Liss, Inc.
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  • 113
    ISSN: 0887-3585
    Keywords: α-zeins ; α-kafirins ; α-coixins ; structure prediction ; gene duplication ; helix packing ; quaternary structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: α-Prolamins are the major seed storage proteins of species of the grass tribe Andropogonea. They are unusually rich in glutamine, proline, alanine, and leucine residues and their sequences show a series of tandem repeats presumed to be the result of multiple intragenic duplication. Two new sequences of α-prolamin clones from Coix (pBCX25.12 and pBCX25.10) are compared with similar clones from maize and Sorghum in order to investigate evolutionary relationships between the repeat motifs and to propose a schematic model for their three-dimensional structure based on hydrophobic membrane-helix propensities and helical “wheels.” A scheme is proposed for the most recent events in the evolution of the central part of the molecule (repeats 3 to 8) which involves two partial intragenic duplications and in which contemporary odd-numbered and even-numbered repeats arise from common ancestors, respectively. Each pair of repeats is proposed to form an antiparallel α-helical hairpin and that the helices of the molecule as a whole are arranged on a hexagonal net. The majority of helices show six faces of alternating hydrophobic and polar residues, which give rise to intersticial holes around each helix which alternate in chemical character. The model is consistent with proteins which contain different numbers of repeats, with oligomerization and with the dense packaging of α-prolamins within the protein body of the seed endosperm. © 1993 Wiley-Liss, Inc.
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  • 114
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 103-107 
    ISSN: 0887-3585
    Keywords: aequorin ; photoprotein ; crystallization ; bioluminescence ; X-ray diffraction ; metal-binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P21212 1; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.
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  • 115
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 116
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 100-102 
    ISSN: 0887-3585
    Keywords: dogfish C-reactive protein ; vapor phase equilibration ; ammonium sulfate ; sitting drop technique ; hexamers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of dogfish (Mustelus canis) C-reactive protein were obtained through vapor phase equilibration using the sitting drop rod technique with ammonium sulfate as the precipitating agent. The space group was determined to be P1 (triclinic lattice) with unit cell dimensions of a = 82.91, b = 92.25 and c = 103.40 Å; α = 83.36°, β = 89.76°, and γ = 81.30°. These crystals diffract to about 2.6 Å resolution and contain two hexamers in the asymmetric unit. © 1993 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
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  • 117
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 108-111 
    ISSN: 0887-3585
    Keywords: ribonuclease H ; Thermus thermophilus HB8 ; DNA/RNA hybrid ; crystallization ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 122 (or P6 522), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley-Liss, Inc.
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  • 118
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. i 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 119
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 113-120 
    ISSN: 0887-3585
    Keywords: thermodynamics ; calorimetry ; protein-hormone interaction ; drug design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the large loss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K. C., Ronco, P. M., Verroust, P. J., Brünger, A. T., Amzel, L. M. Three-dimensional structure of an angiotensin II-Fab complex at 3 Å: Hormone recognition by an anti-idiotypic antibody. Science 257:502-507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30°C the binding is characterized by (1) a ΔH of -8.9 ± 0.7 kcal mol-1, (2) a ΔCp of -240 ± 20 cal K-1 mol-1, and (3) the release of 1.1 ± 0.1 protons per binding site in the pH range 6.0-7.3. Using these values and the previously determined binding constant in phosphate buffer, ΔG at 30°C is estimated as -11 kcal mol-1 and ΔS as 6.9 cal K-1 mol-1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement. Entropically, the loss of configurational entropy is more than compensated by the entropy gain from solvent release associated with the hydrophobic effect. Enthalpically, binding is favored by polar interactions (hydrogen bonding). Consequently, the problem of binding flexible hormones is solved in much the same way as the folding of an unstructured polypeptide chain into a globular protein. © 1993 Wiley-Liss, Inc.
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  • 120
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 133-146 
    ISSN: 0887-3585
    Keywords: coiled coil ; GCN4 ; leucine zipper ; model building ; structure prediction ; molecular dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The recently solved X-ray structure of the dimerization region (“leucine zipper”) of the yeast transcriptional activator GCN4 (O'Shea, E. K., Klemm, J. D., Kim, P. S., Alber, T. Science 254:539-544, 1991) is compared to previously predicted models which had been obtained by a conformational search procedure employing simulated annealing without any knowledge of the crystal coordinates (Nilges, M., Brünger, A. T. Protein Eng. 4:649-659,1991). During the course of the simulated annealing procedure, the models converged towards the X-ray structure. The averaged root mean square difference between the models and the X-ray structure is 1.26 and 1.75 Å for backbone atoms and all nonhydrogen atoms at the dimerization interface, respectively. The local helix-helix crossing angle of the X-ray structure falls within the range predicted by the models; a slight unwinding of the coiled coil toward the N-terminal DNA-binding end of the dimerization region has been correctly predicted. Distance maps between the helices are largely identical. The region around asparagine 20 is asymmetric in the X-structure and in the models. Surface side chain dihedrals showed a large variation in the models although the χ1, χ2, χ3, χ4 3-fold dihedrals were correctly predicted in 69, 42, 43, and 44% of the cases, respectively. Phenomenological free energies of dimerization of the models show little correlation with the root mean square difference between the models and the X-ray structure. © 1993 Wiley-Liss, Inc.
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  • 121
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 15 (1993), S. 121-132 
    ISSN: 0887-3585
    Keywords: epitope ; neuraminidase ; critical contacts ; antigen-antibody complex ; monoclonal antibody ; site-directed mutagenesis ; influenza virus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab1 shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley-Liss, Inc.
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  • 122
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 16 (1993), S. 205-213 
    ISSN: 0887-3585
    Keywords: calcium binding ; crystal structure ; protein stability ; site-directed mutagenesis ; subtilisin ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A version of subtilisin BPN′ lacking the high affinity calcium site (site A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 Å resolution. This protein and the corresponding version containing the calcium A site are describedand compared. The deletion of residues 75-83 was made in the context of four site-specific replacements previously shown to stabilize subtilisin. The helix that in wild type is interrupted by the calcium binding loop, is continuous in the deletion mutant, with normal geometry. A few residues adjacent to the loop, principally those that were involved in calcium coordination, are repositioned and/or destabilized by the deletion. Because refolding is greatly facilitated by the absence of the Caloop, this proteinoffers a new vehicle for analysis and dissection of the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution. © Wiley-Liss, Inc.
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  • 123
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    Proteins: Structure, Function, and Genetics 16 (1993), S. 195-204 
    ISSN: 0887-3585
    Keywords: HIV-1 protease ; HIV-2 protease specificity ; 8-D space ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A vector projection method is proposed to predict the cleavability of oligopeptides by extended-specificity site proteases. For an enzyme with eight specificity subsites the substrate octapeptide can be uniquely expressed as a vector in an 8-dimensional space, whose eight bases correspond to the amino acids at the eight subsites, P1, P1′, P2′, P3′ and P4′, respectively. The component of such a characteristic vector on each of the eight bases is defined as the frequency of an amino acid occurring at a given site. These frequencies were derived from a set of octapeptides known to be cleaved by HIV protease. The cleavability of an octapeptide can then be estimated from the projection of its characteristic vector on an idealized, optimally cleavable vector. The high ratio of correct prediction vs. total prediction for the data in both the training and the testing sets indicates that the new method is self-consistent and efficient. It provides a rapid and accurate algorithm for analyzing the specificity of any multisubsite enzyme for which there is no coupling between subsites. In particular, it is useful for predicting the cleavability of an oligopeptide by either HIV-1 or HIV-2 protease, and hence offers a supplementary means for finding effective inhibitors of HIV protease as potential drugs against AIDS. © Wiley-Liss, Inc.
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  • 124
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    Keywords: ROP ; 4-α-helix bundles ; protein stability ; protein crystallization ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.
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  • 125
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    Proteins: Structure, Function, and Genetics 16 (1993) 
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 126
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    Proteins: Structure, Function, and Genetics 16 (1993), S. 293-300 
    ISSN: 0887-3585
    Keywords: domains ; compactness ; area calculations ; volume calculations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new method for calculating compactness (Z) that uses look-up table-based algorithms for area and volume computations is introduced. These algorithms can be used in any iterative area orvolume calculation, are more than 1000 times faster than the originalalgorithms, and have equal or better precision. With the faster algorithms it is now possible to calculate the compactness of all continuous units in a protein, and to precisely locate the optimal compact units without the screening functions and limited resolution used previously. These methods have been incorporated into a fully automatic domain finding algorithm, and this method has been applied to the 21 proteins originally analyzed as well as 12 additional proteins. This method is robust, and yields similar units even when applied to coordinates of protein crystals grown under different experimental conditions. © 1993 Wiley-Liss, Inc.
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  • 127
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    Proteins: Structure, Function, and Genetics 16 (1993), S. 172-194 
    ISSN: 0887-3585
    Keywords: serine protease ; active site solvation ; stochastic simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The motions of water molecules, the acyl moiety, the catalytic triad, and the oxyanion binding site of acyl-chymotrypsin were studied by means of a stochastic boundary molecular dynamics simulation. A water molecule that could provide the nucleophilic OH- for the deacylation stage of the catalysis was found to be trapped between the imidazole ring of His-57 and the carbonyl carbon of the acyl group. It makes a hydrogen bond with the Nε2 of His-57 and is heldin place through a network of hydrogen-bonded water molecules in theactive site. The water molecule was found as close as 2.8 Å to the carbonyl carbon. This appears to be due to the constraints imposed by nonbonded interaction in the active site. Configurations were found in which one hydrogen of the trapped water shared a bifurcated hydrogen bond with His-57-Nε2 and Ser-195-0γ with the water oxygen very close to the carbonyl carbon. The existence of such a water molecule suggests that large movement of the His-57 imidazole ring between positions suitable for providing general-base catalyzed assistance and for providing general-acid catalyzed assistance may notbe required during the reaction. The simulation indicates that the side chains of residues involved in catalysis (i.e., His-57, Ser-195, and Asp-102) are significantly less flexible than other side chains in the protein. The 40% reduction in rms fluctuations is consistent with a comparable reduction calculated from the temperature factors obtained in the X-ray crystal-lographic data of γ-chymotrypsin. The greater rigidity of active site residues seems to result from interconnected hydrogen bonding networks among the residues and between the residues and the solvent water in the active site. © Wiley-Liss, Inc.
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  • 128
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    Proteins: Structure, Function, and Genetics 17 (1993), S. 337-346 
    ISSN: 0887-3585
    Keywords: RNase H ; Mg2+-binding site ; catalytic mechanism ; molecular replacement ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: To obtain more precise insight into the Mg2+-binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg2+. The analyzed cocrystal, which is not isomorphous with the Mg2+-free crystal previously refined at 1.48 Å resolution, was grown at a high MgSO4 concentration more than 100 mM so that even weakly bound Mg2+ sites could be identified. The structure was solved by the molecular replacement method, using the Mg2+-free crystal structure as a search model, and was refined to give a final R-value of 0.190 for intensity data from 10 to 2.8 Å, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg2+-bound and the metal-free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg2+ atom located at a position almost identical to that previously found by the soaking method. Although the two metal-ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990) and partially supported by the crystallographic study of inactive HIV-1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88-95, 1991), the present result excludes the possibility that RNase HI requires two metal-binding sites for activity. In contrast to the features in the metal-free enzyme, the side chains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg2+ in the metal-bound crystal. © 1993 Wiley-Liss, Inc.
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  • 129
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    Proteins: Structure, Function, and Genetics 17 (1993), S. 355-362 
    ISSN: 0887-3585
    Keywords: protein folding ; protein modeling ; molecular force fields ; protein structure determination ; Boltzmann's principle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A major problem in the determination of the three-dimensional structure of proteins concerns the quality of the structural models obtained from the interpretation of experimental data. New developments in X-ray crystallography and nuclear magnetic resonance spectroscopy have acceleratedd the process of structure determination and the biological community is confronted with a steadily increasing number of experimentally determined protein folds. However, in the recent past several experimentally determined protein structures have been proven to contain major errors, indicating that in some cases the interpretation of experimental data is difficult and may yield incorrect models. Such problems can be avoided when computational methods are employed which complement experimental structure determinations. A prerequisite of such computational tools is that they are independent of the parameters obtained from a particular experiment. In addition such techniques are able to support and accelerate experimental structure determinations. Here we present techniques based on knowledge based mean fields which can be used to judge the quality of protein folds. The methods can be used to identify misfolded structures as well as faulty parts of structural models. The techniques are even applicable in cases where only the Cα trace of a protein conformation is available. The capabilities of the technique are demonstrated using correct and incorrect protein folds. © 1993 Wiley-Liss, Inc.
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  • 130
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    Proteins: Structure, Function, and Genetics 17 (1993), S. 363-374 
    ISSN: 0887-3585
    Keywords: sequence analysis ; homology search ; ANK repeat ; horizontal gene transfer ; cell cycle proteins ; transcription factor NF-κB ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Based on pattern searches and systematic database screening, almost 650 different ankyrin-like (ANK) repeats from nearly all phyla have been identified; more than 150 of them are reported here for the first time. Their presence in functionally diverse proteins such as enzymes, toxins, and transcription factors strongly suggests domain shuffling, but their occurrence in prokaryotes and yeast excludes exon shuffling. The spreading mechanism remains unknown, but in at least three cases horizontal gene transfer appears to be involved. ANK repeats occur in at least four consecutive copies. The terminal repeats are more variable in sequence. One feature of the internal repeats is a predicted central hydrophobic α-helix, which is likely to interact with other repeats. The functions of the ankyrin-like repeats are compatible with a role in protein-protein interactions. © 1993 Wiley-Liss, Inc.
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  • 131
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    Proteins: Structure, Function, and Genetics 17 (1993), S. 391-411 
    ISSN: 0887-3585
    Keywords: protein sequence ; sequence repeats ; sequence alignments ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: An automated algorithm is presented that delineates protein sequence fragments which display similarity. The method incorporates a selection of a number of local nonoverlapping sequence alignments with the highest similarity scores and a graphtheoretical approach to elucidate the consistent start and end points of the fragments comprising one or more ensembles of related subsequences. The procedure allows the simultaneous identification of different types of repeats within one sequence. A multiple alignment of the resulting fragments is performed and a consensus sequence derived from the ensemble(s). Finally, a profile is constructed form the multiple alignment to detect possible and more distant members within the sequence. The method tolerates mutations in the repeats as well as insertions and deletions. The sequence spans between the various repeats or repeat clusters may be of different lengths. The technique has been applied to a number of proteins where the repeating fragments have been derived from information additional to the protein sequences. © 1993 Wiley-Liss, Inc.
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  • 132
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    Biotechnology and Bioengineering 41 (1993), S. 1-1 
    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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  • 133
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    Biotechnology and Bioengineering 41 (1993), S. 35-42 
    ISSN: 0006-3592
    Keywords: pattern recognition ; machine vision ; tissue cultures ; Betula pendula Roth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendula Roth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed. © 1993 John Wiley & Sons, Inc.
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  • 134
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    Biotechnology and Bioengineering 41 (1993), S. 43-54 
    ISSN: 0006-3592
    Keywords: acetic acid ; alkaline protease ; Bacilus firmus ; continuous culture ; extracellular enzymes ; carbon/nitrogen/phosphorus limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. © 1993 John Wiley & Sons, Inc.
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  • 135
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    Biotechnology and Bioengineering 41 (1993), S. 95-103 
    ISSN: 0006-3592
    Keywords: enzymatic esterification ; equilibrium ; log P ; organic solvent choice ; lipase ; two-phase system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of organic solvents on the equilibrium position of lipase-catalyzed esterification of glycerol and decanoic acid has been investigated. The reaction is carried out in an aqueous-organic two-phase system. In polar solvents, high mole fractions of monoacylglycerol and low mole fractions of triacylglycerol and measured, while in nonpolar solvents, the measured differences in the mole fractions of monodi-, and triacylglycerols are less. There is a good correlation between the ester mole fractions at equilibrium and the log P of the solvent (partition coefficient in n-octanolwater), however, only if the group of tertiary alcohols is excluded. In the plot of the easter mole fractions as a function of the logarithm of hte solubility of water in the organic solvent, the tertiary alcohols can be included; however, in this case other deviations appear.For the prediction of the effect of organic solvents on the ester mole fractions at reaction equilibrium in nondilute reaction systems with a water activity below 1, the program TREP (Two-phase Reaction Equilibrium Prediction) is developed, which is based on the UNIFAC group contribution method. With this model the equilibrium data are essentially predicted from basic thermodynamic data. The required equilibrium constants are estimated from experiments without an organic solvent in the reaction medium. The mole fractions calculated by TREP show the same trends as the experimentally measured mole fractions; however, some variation is observed in the absolute values. These deviations may be due to inaccuracies in the UNIFAC group contribution method. TREP is found to be a correct method to predict within some limits the ester mole fractions at equilibrium for all mixtures of solvents, substrates, and products. The production of monoester can be enhanced in reaction system with a sufficient high concentration of a polar solvent. In experiments with a triglymeto-decanoic acid ratio of 5, almost no di-and triesters can be detected at equilibrium. © 1993 John Wiley & Sons, Inc.
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  • 136
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    Biotechnology and Bioengineering 41 (1993), S. 134-147 
    ISSN: 0006-3592
    Keywords: displacement ; elution ; optimization ; preparative chromatography ; production rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The results of a study of the optimization of the experimental conditions for maximum production rate in overloaded elution and displacement chromatography are discussed. This study is based on the use of the equilibrium-dispersive model of chromatography and the competitive Langmuir isotherms to calculate individual band profiles in the elution and displacement modes, and of a simplex algorithm to optimize the production rate. The operating parameters (sample size, mobile phase velocity, and the displacer concentration in the displacement model) and the column design parameters (column length and average particle diameter) are optimized simultaneously. Binary mixtures having relative concentrations 3:1 and 1:3, and separation factors of 1.2 to 1.8 are investigated. One of our major results is that, in both modes of chromatography, the maximum production rate is achieved at very low values of the retention factors, k′, much lower than those used in current practice. In all cases, unless k′ exceeds greatly that optimum value, the production rate is higher in overloaded elution than in displacement chromatography. This is particularly true for the extraction of a minor component, which is eluted second. © 1993 John Wiley & Sons, Inc.
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  • 137
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    Biotechnology and Bioengineering 41 (1993), S. 316-324 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; acetic acid ; inhibition ; glycine ; methionine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more β-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h-1, the methionine addition enhanced β-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h -1, the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. © 1993 John Wiley & Sons, Inc.
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  • 138
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    Biotechnology and Bioengineering 41 (1993), S. 330-340 
    ISSN: 0006-3592
    Keywords: hybridoma ; Immobilization ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (qMAb) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the qMAb with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/ϒ2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T- and spinner flasks in order to determine whether cultivation methods (T- and spinner flasks) and bead size influence the qMAb Free-suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the qMab of the entrapped cells are reversible.The cultivation methods was found to influence significantly the qMAb of the entrapped cells. When the entrapped cells in 1-mn diameter beads were cultivated in T-flasks, the qMAb was not increased by 200% as previously observed in an entrapped cell culture using 1-mm-diameter alginate beads in spinner flasks. The qMAb of the entrapped cell was approximately 58% higher than that of the free-suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1- to 3-mm diameter did not significantly influence the qMAb, regardless of cultivations methods. The changes in qMAb of an entrapped cells were reversible. When the free-suspended cells recovered from the T- and spinner flasks were sub-cultured in T- and spinner flasks enhanced qMAb of the entrapped cells in both cases decreased to the level of the free-suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the qMAb. © 1993 John Wiley & Sons, Inc.
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  • 139
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    Biotechnology and Bioengineering 41 (1993), S. 380-389 
    ISSN: 0006-3592
    Keywords: density-dependent growth ; anchorage-dependent cells ; image analysis ; CHO cells ; model simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The density-dependent growth of Chinese hamster ovary (CHO) cells was monitored on-line by using an inverted microscope. A flow system was employed for cell cultivation so that nutrient concentration could be maintained and metabolic wastes were removed. With the help of video image analysis, local cells density could be accurately calculated and cell motility and exposed cell surface area could be estimated. A computer program which accounted for change of sell size and translocation of cells was developed to stimulate dell growth. The stimulated results of the population dynamics and the variations in cell size showed good agreement with our experimental observations, Cell motility and initial cell distribution on the substratum were found to have strong effect on cell growth. © 1993 John Wiley & Sons, Inc.
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  • 140
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    Biotechnology and Bioengineering 41 (1993), S. 390-393 
    ISSN: 0006-3592
    Keywords: organic solvents ; enzyme catalysis ; immiscibility with water ; hydrophobicity of solvents ; dipole moment dielectric constant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The question of whether the solvent's water-immiscibility is relevant to enzymatic activity was addressed by assaying four different hydrolases (three lipases and one protease) in nine anhydrous solvents of similar hydrophobicities of which four were infinitely miscible with water and five were not. For no enzyme was a jump in activity observed upon a transition from water-miscible to water-immiscible solvent. The relevance of solvent apolarity to enzymatic efficiency was also examined. To this end, three groups of isomeric anhydrous solvents were selected where within each group of isomeric anhydrous solvents were selected where within each group one solvent was apolar (i.e., lacked a permanent dipole moment). For none of the four enzymes studied was activity significantly higher in apolar solvents than in their polar counterparts. Thus we conclude that often-cited solvent's immiscibility with water and apolarity by themselves are irrelevant to enzymatic activity. © 1993 John Wiley & Sons, Inc.
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  • 141
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    Biotechnology and Bioengineering 41 (1993), S. 422-428 
    ISSN: 0006-3592
    Keywords: entropy of growth ; Escherichia coli K-12 ; entropy of anabolism ; entropy change ; entropy of formation ; entropy of formation of cells ; cellular entropy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ΔSf′ of one unit carbon formula weight of Escherichia coli K-12 cells, when grown on succinic acid, was calculated to be -80.13 J/deg. This value could then be used to calculate the entropy change accompanying the anabolism and metabolism of succinic acid to be 30.82 J/deg and 32.40 J/mol deg, respectively. The entropy of one unit carbon formula weight of dried E. Coli K-12 cells is calculated to be 94.40 J/deg, which when divided by the mass of these cells becomes 3.90 J/g deg. The corresponding entropy of succinic acid is 2.77 J/g deg, making it apparent that the entropy per unit mass of the cells is greater than that of the substrate. It might be thought that because the cells appear to be so much more complex than the substrate, the cells should have a lesser entropy per unit mass than the substrate. That this does not appear to be true leads to the conclusion that the macromolecular organization (informational content?) of the cells contributes only in a very minor way to the total physical entropy of cells. © 1993 John Wiley & Sons, Inc.
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  • 142
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    Biotechnology and Bioengineering 41 (1993), S. 451-458 
    ISSN: 0006-3592
    Keywords: immobilized glucose isomerase ; substrate protection ; reactor analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of substrate protection on enzyme deactivation was studied in a differential bed and a packed bed reactor using a commercial immobilized glucose isomerase (Swetase, Nagase Co.). Experimental data obtained from differential bed reactor were analyzed based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrate was considered. The deactivation constant of the enzyme-substrate complex was found to be about half of that of the free enzyme. The mathematical analysis describing the performance of a packed bed reactor under the considerations of the effects of substrate protection, diffusion resistance, and enzyme deactivation was studied. The system equations for the packed bed reactor were solved using an orthogonal collocation method. The presence of substrate protection and the diffusion effect within the enzyme particles resulted in an axial variation of effectiveness factor, ηD, along the length of the packed bed. The axial distribution profile of ηD was found to be dependent on the operation temperature, Based on the effect of substrate protection, a better substrate feed policy could be theoretically found for promoting productivity in long-term operation. © 1993 John Wiley & Sons, Inc.
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  • 143
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    Biotechnology and Bioengineering 41 (1993), S. 489-492 
    ISSN: 0006-3592
    Keywords: reverse micelles ; back-extraction ; silica ; proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative “back extraction” procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (α-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a α-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. © 1993 John Wiley & Sons, Inc.
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  • 144
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    Biotechnology and Bioengineering 41 (1993), S. 512-524 
    ISSN: 0006-3592
    Keywords: biofiltration ; biofilter modeling ; methanol ; biodegradation ; VOC emissions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biofiltration of solvent and fuel vapors may offer a costeffective way to comply with increasingly strict air emission standards. An important step in the development of this technology is to derive and validate mathematical models of the biofiltration process for predictive and scaleup calculations. For the study of methanol vapor biofiltration, an 8-membered bacterial consortium was obtained from methanol-exposed soil. The bacteria were immobilized on solid support and packed into a 5-cm-diameter, 60-cm-high column provided with appropriate flowmeters and sampling ports. The solid support was prepared by mixing two volumes of peat with three volumes of perlite particles (i.e., peat-perlite volume ratio 2:3). Two series of experiments were performed. In the first, the inlet methanol concentration was kept constant while the superficial air velocity was varied from run to run. In the second series, the air flow rate (velocity) was kept constant while the inlet methanol concentration was varied. The unit proved effective in removing methanol at rates up to 112.8 g h-1 m-3 packing. A mathematical model has been derived and validated. The model described and predicted experimental results closely. Both experimental data and model predictions suggest that the methanol biofiltration process was limited by oxygen diffusion and methanol degradation kinetics. © 1993 John Wiley & Sons, Inc.
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  • 145
    ISSN: 0006-3592
    Keywords: Continuous Culture ; two-liquid-phase system ; recombinant E. coli-alk system ; bioconversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Escherichia coli is able to grow on sugars in the presence of a bulk n-alkane phase. When E. coli is equipped with the alk genes from Pseudomonas oleovorans, the resulting recombinant strain converts n-alkanes into the corresponding alkanoic acids. To study the effects of growth rate and exposure to a bulk apolar phase on the physiology and the productivity of E. coli, we have grown this microorganism in two-liquid-phase continuous cultures containing 5% (v/v) n-octane.In contrast to batch cultures of wild-tape E. coli grown in the presence of n-octane, cells remained viable during the entire continuous culture, which lasted 200 h. Bioconversion of n-octane to n-octanoic acid by a recombinant E. coli (alk+) in a two-liquid-phase continuous culture was made possible by optimizing both the recombinant host strain and the conditions of culturing the organism. Continuous production in such two-phase systems has been maintained for the least 125 h without any changes in the product concentration in the fermentation medium. The volumetric productivity was determined as a function of growth rate and showed a maximum at a dilution rate D = 0.32 h-1, reaching a continuous production rate of 0.5 g octanoate/L · h (4 tons/m3 · year). © 1993 John Wiley & Sons, Inc.
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  • 146
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    Biotechnology and Bioengineering 41 (1993), S. 287-295 
    ISSN: 0006-3592
    Keywords: carboxylic and amino acids ; supported ; emulsion ; hybrid liquid membranes ; facilitated transport ; uphill pumping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Liquid-liquid extraction and membrane separation are well-known separation method of extensive industrial application. Their incorporation into liquid membranes has the potential of several advantages, some of which are of particular interest for the recovery of carboxylic and amino acids: selectivities higher than those attainable by current separation methods, saving on energy costs for final concentration of separated products, high fluxes, compact installation, and low capital and operation costs. Stability of the liquid advantages, can be secured by utilizing extractant blocking polymeric membranes, Applicability, process consideration, and economic implications for recovery for carboxylic and amino acids by various extractant/membrane combinations are discussed. © 1993 John Wiley & Sons, Inc.
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  • 147
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    Biotechnology and Bioengineering 41 (1993), S. 599-602 
    ISSN: 0006-3592
    Keywords: optimal control ; iterative dynamic programming ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: By using penalty functions to handle state constraints, iterative dynamic programming can be used in a straightforward manner for the optimization of fedbatch fermentors. No computational difficulties were encountered and better results are obtained than previously reported in the literature for a fed-batch fermentor for biosynthesis of penicillin. © 1993 Johy Wiley & Sons, Inc.
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  • 148
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    Biotechnology and Bioengineering 41 (1993), S. 617-624 
    ISSN: 0006-3592
    Keywords: crossflow filtration ; microfiltration ; baker's yeast ; Saccharomyces cerevisiae ; molasses ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Factors affecting the performance of crossflow filtration were investigated with a thin-channel module and yeast cells. In crossflow filtration of Saccharomyces cerevisiae cells cultivated with YPD medium (Yeast extract, polypeptone, and dextrose) and suspended in saline, a steady state was attained within several minutes when the cell concentration was low and the circulation flow rate was high. The steady-state flux and the change in flux during the initial unsteady state were explained well by conventional filtration theory, with the amount of cake deposited and the mean specific resistance to the cake measured in a dead-end filtration apparatus used in calculation. When the circulation flow rate was lower than a critical value, a part of the channel of the crossflow filtration module was plugged with cell cake, and thus the steady-state flux was low. In crossflow filtration of suspensions of commercially available baker's yeast, the flux gradually decreased, and the flux after 8 h of filtration was lower than the value calculated by filtration theory. Fine particles contaminating the baker's yeast was responsible for the decrease. A similar phenomenon was responsible for the decrease. A similar phenomenon was observed in crossflow filtration of a broth of S. cerevisiae cells cultivated in molasses medium, which also contains such particles, had no effect of the permeation flux during crossflow filtration. © 1993 John Wiley & Sons, Inc.
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  • 149
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    Biotechnology and Bioengineering 41 (1993), S. 654-658 
    ISSN: 0006-3592
    Keywords: polyethylene glycol ; hydrophobicity ; enzymatic synthesis ; cephalexin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K7-ADCA in the presence of PEG was smaller than that in the control system (without PEG addition). © 1993 John Wiley & Sons, Inc.
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  • 150
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    Biotechnology and Bioengineering 41 (1993) 
    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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  • 151
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    Biotechnology and Bioengineering 41 (1993), S. 707-714 
    ISSN: 0006-3592
    Keywords: genetically structured mathematical model ; trp operon ; cloned gene expression control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A genetically structured mathematical model of the trp operon based on known molecular interactions of aporepressor, corepressor, and inducer is proposed. The model simulates, both qualitatively and quantitatively, the influence of these regulatory species on the extent of repression and expression of cloned gene products. It shows that at low aporepressor concentration, full repression is not possible even with high tryptophan levels, resulting in leaky expression. Calculations based on the model enabled predictions of optimum levels of aporepressor and tryptophan for effective repression and, concurrently, the β-indoleacrylic acid concentrations required for induction for both low and high plasmid copy number clones. Using the model we attempted to provide explanations for seemingly anomalous and sometimes contradictory observations by researchers when working with the trp promoter. © 1993 John Wiley & Sons, Inc.
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  • 152
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    Biotechnology and Bioengineering 41 (1993), S. 736-744 
    ISSN: 0006-3592
    Keywords: lactose ; effective diffusivity ; acidogenic biofilm ; biofilm void fraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Effective diffusivity of lactose in active acidogenic biofilms was measured at 35°C and pH 4.6 with a specially designed diffusion cell. The diffusion cell was designed and operated in such a way that the lactose concentrations on the surface and at the center of a living bacterial aggregate could be measured at steady state. As a model parameter in a widely accepted reaction-diffusion equation which describes lactose distribution in living biofilms, the effective diffusivity of lactose in the biofilms was found to be about 65% of the lactose diffusivity in free solutions. It was experimentally determined that the active biofilms had about 66% void volume made up of channels through which the lactose molecules were transported into the bacterial aggregates. Therefore, the decrease in lactose diffusivity was mainly caused by the biofilm's solid biomass fraction rather than the tortuosity of the channels. © 1993 John Wiley & Sons, Inc.
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  • 153
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    Biotechnology and Bioengineering 41 (1993), S. 761-770 
    ISSN: 0006-3592
    Keywords: ultrafiltration membrane bioreactor ; reversed micelles ; lipase ; product separation ; lipolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. © 1993 Wiley & Sons, Inc.
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  • 154
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    Biotechnology and Bioengineering 41 (1993), S. 411-421 
    ISSN: 0006-3592
    Keywords: bacterial cytochrome P-450cam ; hydrocarbon fermentation ; halocarbon degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cytochrome P-450cam monooxygenase is an important bacterial redox enzyme system with potential commercial value for detoxifying trace hydrocarbon contaminants, catalyzing regiospecific hydroxylations, and amperometric biosensing. The present study was undertaken to increase productivity of this enzyme, which is induced in its host, pseudomonas putida PpG 786, by D(+)-camphor. Culture processes were studied in batch, fed-batch, and continuous modes to evaluate metabolic behavior and develop constitutive equations for specific rate of growth (μ), camphor utilization (qp). Fed-batch culture was characterized by an extended linear growth phase which is often encountered in hydrocarbon fermentations. Inhibition by the camphor solvent, dimethylformamide, was assessed. Production of the terminal protein of the p-450cam enzyme system, cytochrome m, was shown to depend on growth medium iron content in fed-batch culture and was increased by 130% over previously protocols by eliminating iron deficiency. A continuous process that enables greater production rates was developed by using oxygen enrichment while simultaneously reducing gas throughput. Camphor and oxygen requirements were determined for fedbatch and continuous growth. © 1993 John Wiley & Sons, Inc.
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  • 155
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    Biotechnology and Bioengineering 41 (1993), S. 459-464 
    ISSN: 0006-3592
    Keywords: Thiobacillus ferrooxidans ; carbon dioxide uptake ; carbon dioxide inhibition ; bacterial leaching ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of carbon dioxide concentration on the bacterial leaching of a pyrite-arsenopyrite ore concentrate was studied in continuous-flow reactors. Steady-state operation with two feed slurry densities, 6 wt% and 16 wt% solids, were tested for the effect of carbon dioxide concentration. Bacterial growth rates were estimated via the measurement of carbon dioxide consumption rates. Aqueous-phase carbon dioxide concentrations in excess of 10 mg/L were found to be inhibitory to bacterial growth. © 1993 John Wiley & Sons, Inc.
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  • 156
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    Biotechnology and Bioengineering 41 (1993) 
    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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  • 157
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    Biotechnology and Bioengineering 41 (1993), S. 531-540 
    ISSN: 0006-3592
    Keywords: horseradish peroxidase ; reversed micelles ; phenolic polymers ; enzyme kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The enzyme horseradish peroxidase, when encapsulated in reversed micelles, is capable of catalyzing the synthesis of phenolic and aromatic amine polymers. The synthesis of polyethylphenol is specifically considered in this article and is found to be extremely feasible in the micellar system. Polymer chain growth can be controlled to some degree by manipulating the ability of the solvent to sustain chain solubility; this is effectively done by adjusting the surfactant concentration. This results in a degree of control of polymer molecular weight. The synthesized polymer drops out of solution and can be easily recovered. © 1993 John Wiley & Sons, Inc.
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  • 158
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    Biotechnology and Bioengineering 41 (1993), S. 572-580 
    ISSN: 0006-3592
    Keywords: phenol degradation ; continuous culture ; Pseudomonas putida ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h-1 and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l-1. These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. © 1993 John Wiley & Sons, Inc.
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  • 159
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    Biotechnology and Bioengineering 41 (1993), S. 957-963 
    ISSN: 0006-3592
    Keywords: concanavalin A ; soluble protein oligomer ; insulin derivatives ; glucose binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Concanavalin A, (Con A, MW 26,500/monomer unit) was crosslinked with glutaraldehyde to form soluble, high-molecular-weight (larger than MW 300,000) Con A Oligomers. After filtration to remove insoluble and low-molecular-weight portions (below 300,000 daltons), the size and molecular-weight distribution were characterized by laser light scattering and gel-filtration chromatography. The molecular-size determined by laser light scattering ranged from 870 to 4070 Å, while the molecular weight determined by gel chromatography ranged from 6 × 105 to higher than 2 × 106 daltons. The affinity and kinetics of Con A oligomer binding to polysaccharide (glycogen) were evaluated by precipitation test and turbidity development, respectively. The binding with glycogen was strongest at neutral pH and showed similar activity to unmodified Con A molecules. The binding constants of α-D-glucose and succinyl-aminophenyl α-D glucopyranoside-insulin to Con A oligomer were 1.0 × 103M-1 and 4.5 × 104M-1, respectively and the binding capacity of the oligomer was nearly 85% to 95% of monomeric Con A. The complexes of saccharides and soluble Con A oligomer were stable for at least 7 days. © 1993 Wiley & Sons, Inc.
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  • 160
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    Biotechnology and Bioengineering 41 (1993), S. 991-997 
    ISSN: 0006-3592
    Keywords: protein purification ; affinity precipitation ; avidin ; biotin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel technique for affinity precipitation has been developed in which multimeric target proteins are precipitated as a result of network formation by polymer-conjugated ligands (polyligands). A polyligand precipitant for avidin was synthesized by conjugation of biotin to a polyacrylamide-based backbone. The effects of mixing conditions, ligand substitution frequency, and molecular weight on affinity precipitation were examined using the biotin-PAAm precipitant. Biotin was replaced by iminobiotin to study the effect of the ligand-protein dissociation constant o affinity precipitation. The iminobiotin-PAAm precipitant was also used to examine the reversibility of the precipitation and recovery of the target protein after precipitation. © 1993 Wiley & Sons, Inc.
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  • 161
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    Biotechnology and Bioengineering 41 (1993), S. 1007-1013 
    ISSN: 0006-3592
    Keywords: pressure drop ; solid state fermentation ; Aspergillus niger ; ion exchange resin ; permeability ; wheat bran ; cane Bagasse ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The measurement of pressure drop(ΔP) across an aerated fermentation bed is proposed as alternative on-line sensor for the qualitative and, in some cases, quantitative, macroscopic changes in a static solid state fermentor. An increase in the ΔP is correlated with the evolution of the different phases of Aspergillus niger growth: germination, vegetative growth, limitation, and sporulation, we observed in the microscope. For the case where the support is not modified during the fermentation and the water content remains constant, i.e., a synthetic resin (Amberlite IRA-900), the gas phase permeability of the bed is directly related to the biomass content. For example, the permeability of the bed is reduced to 5% of the initial value when biomass attains 21 mg dry biomass/g dry support. Biomass was appropriately predicted from the ΔP measurements in an independent test. Experiments with different initial sucrose solution concentrations showed that biomass could not be produced beyond a certain level (21.5 mg dry biomass/g dry support) which suggests steric limitations. For the case of wheat bran and cane bagasse, the increase in ΔP was related qualitatively to the evolution in the growth and the morphology of the mold . © 1993 Wiley & Sons, Inc.
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  • 162
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    Biotechnology and Bioengineering 41 (1993), S. 1039-1047 
    ISSN: 0006-3592
    Keywords: polyethylene glycol ; albumin ; ultrafiltration ; separation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Transmission of polyethylene glycol (PEG) through ultrafiltration membranes has been studied under various operating conditions of pressure, crossflow, and concentration, using different membranes cut-offs and two module designs with the aim of understanding the separation of PEG from BSA. The influence of protein adsorption and fouling of the choice of a membrane has also been considered. Retention depends in general on the molecule to average pore size ratio, as expected, but also on concentration polarization. Accordingly, all operating and design parameters favoring concentration polarization lead to higher transmission. At high fluxes, flexible macromolecules can pass through the membrane, even if the random coil is larger than the apparent average pore. From a process selectivity point of view, the best way to separate PEG from BSA would be to use a membrane totally retaining BSA and to enhance concentration polarization of PEG. Unfortunately, such conditions also increase fouling and concentration polarization by BSA, which limits flux and thus PEG concentration polarization and transmission. Consequences of such conditions on separation efficiency are discussed. © 1993 Wiley & Sons, Inc.
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  • 163
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    Biotechnology and Bioengineering 41 (1993), S. 1082-1091 
    ISSN: 0006-3592
    Keywords: waste-activated sludge ; two-phase digestion ; sludge solubilization ; biopolymer hydrolysis ; kinetic model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The anaerobic bioconversion of raw and mechanically lysed waste-activated sludge was kinetically investigated. The hydrolysis of the biopolymers, such as protein, which leaked out from the biological sludge with ultrasonic lysis, was a first-order reaction in anaerobic digestion and the rate constant was much higher that the decay rate constant of the raw waste activated sludge. An anaerobic digestion model that is capable of evaluating the effect of the mechanical sludge lysis on digestive performance was developed. The present model includes four major biological processes-the release of intracellular matter with sludge lysis; hydrolysis of biopolymers to volatile acids; the degradation of various volatile acids to acetate; and the conversion of acetate and hydrogen to methane. Each process was assumed to follow first order kinetics. The model suggested that when the lysed waste-activated sludge was fed, the overall digestive performance remarkably increased in the two-phase system consisting of an acid forming process and a methanogenic process, which ensured the symbiotic growth of acetogenic and methanogenic bacteria. © 1993 Wiley & Sons, Inc.
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  • 164
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    Biotechnology and Bioengineering 41 (1993), S. 1112-1120 
    ISSN: 0006-3592
    Keywords: luminol chemiluminescence ; peroxidase ; hydrogen peroxide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. © 1993 Wiley & Sons, Inc.
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  • 165
    ISSN: 0006-3592
    Keywords: unsteady state ; kinetic parameters ; Pichia stipitis ; D-xylose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D-xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, μm and Ks can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of Ks and μm and a little over 4% for μm were the deviations under the latter ones. © 1993 Wiley & Sons, Inc.
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  • 166
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    Biotechnology and Bioengineering 42 (1993), S. 30-36 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; fiber optic ; firefly luciferase ; on-line ; viability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 106 cells/mL, or an OD600 of 0.004, are easily detectable and concentrations greater than 1010 cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. © 1993 John Wiley & Sons, Inc.
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  • 167
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    Biotechnology and Bioengineering 42 (1993), S. 59-73 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; amino acids ; nucleotides ; biosynthesis ; linear optimization ; metabolic fluxes ; metabolic engineering ; stoichiometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. © 1993 John Wiley & Sons, Inc.
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  • 168
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    Biotechnology and Bioengineering 42 (1993), S. 103-110 
    ISSN: 0006-3592
    Keywords: lipases ; selectivity ; esterifications ; microemulsions ; reverse micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. © 1993 John Wiley & Sons, Inc.
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  • 169
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    Biotechnology and Bioengineering 42 (1993), S. 125-132 
    ISSN: 0006-3592
    Keywords: steam-in-place sterilization ; dead-ended tube ; dead-legs ; Bacillus stearothermophilus ; steam sterilization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Effect of tube diameter on steam-in-place sterilization of dead-ended tubes was studied by examining temperature profiles and rates of kill of Bacillus stearothermophilus spores. Time required for sterilization was determined for 9.4-cm-long tubes with various inside diameters from 0.4 to 1.7 cm. Sterilization time increased with decreasing tube diameter. Experimentally measured kill kinetics in 1.7-cm tubes were in agreement with those predicted if measured temperatures represented saturated steam. A 12-log spore reduction was achieved in 1.7-cm diameter vertical and horizontal tubes in less than 63 minutes. For smaller diameter tubes, entrapped air remained after 2 hours and rates of kill were very dependent on position within the tube, tube diameter, and tube orientation with respect to the gravitational vector. Times to achieve a 1-log drop in spore population in the smaller tubes were as much as 10 times greater than those expected if measured temperatures represented saturated steam. Sterilization was not achieved throughout the 0.4-cm tubes. Recommendations are made for including steam bleeders or using prevaccum cycles for these smaller diameter tubes. © 1993 John Wiley & Sons, Inc.
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  • 170
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    Biotechnology and Bioengineering 42 (1993), S. 159-166 
    ISSN: 0006-3592
    Keywords: anaerobic digestion ; ammonia inhibition ; manure ; mathematical model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model for anaerobic degradation of complex organic material, such as manure, has been developed. The model includes an enzymatic hydrolytic step and four bacterial steps and involves 12 chemical compounds. The model focuses on ammonia inhibition and includes a detailed description of pH and temperature characteristics in order to accurately simulate free ammonia concentration. Free ammonia and acetate constitute the primary modulating factors in the model. The model has been applied for the simulation of digestion of cattle manure in continuously stirred tank reactors (CSTRs), and results compare favorably with experimental data. © 1993 John Wiley & Sons, Inc.
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  • 171
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    Biotechnology and Bioengineering 42 (1993), S. 178-184 
    ISSN: 0006-3592
    Keywords: recombinant β-galactosidase fusion protein ; chelating peptide ; immobilized metal affinity chromatography ; immobilized enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The strong interaction of hexa-histidine fusion proteins with metal chelate adsorbents was utilized to immobilize β-galactosidase with a hexa-histidine peptide at the N-terminus to the Ni2+-nitrilotriacetic acid adsorbent. The fusion protein was cloned and expressed in Escherichia coli. The purified soluble fusion protein showed the same specific activity as the purified β-galactosidase and retained 64 percent of its β-galactosidase activity when bound to the adsorbent. To demonstrate the potential of the immobilized β-galactosidase in organic chemistry, allyl-β-D-galactosidase was synthesized from lactose and allyl alcohol on a gram scale. The same enzyme preparation was reused in three subsequent batches to prepare the model compound with high yield. © 1993 John Wiley & Sons, Inc.
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  • 172
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    Biotechnology and Bioengineering 42 (1993), S. 222-234 
    ISSN: 0006-3592
    Keywords: segregated modeling ; plasmid distribution ; plasmid stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a “downward selective pressure,” is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. © 1993 John Wiley & Sons, Inc.
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  • 173
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    Biotechnology and Bioengineering 42 (1993), S. 50-58 
    ISSN: 0006-3592
    Keywords: emulsion liquid membrane ; lactic acid ; organic acid recovery ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n-heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. © 1993 John Wiley & Sons, Inc.
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  • 174
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    Biotechnology and Bioengineering 42 (1993), S. 279-283 
    ISSN: 0006-3592
    Keywords: image analysis ; UASB digester granules ; sizing ; density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two-dimensional image analysis was applied to counting, sizing, and density determinations of granules in full-scale and laboratory-scale upflow anaerobic sludge blanket (UASB) digesters. An advantage of this technique for monitoring laboratory-scale digester sludge is the small amount of material required for analysis. Quantification of number of granules using this method correlated well with dry weight determinations (r = 0.989). Distinguished granule size increased with time throughout the digestion process, supported by dry weight determinations which indicated an increase in biomass. The monitoring of granule density may reveal subtleties of the selection pressure placed on granules not noticed previously. © 1993 John Wiley & Sons, Inc.
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  • 175
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    Biotechnology and Bioengineering 42 (1993), S. 303-308 
    ISSN: 0006-3592
    Keywords: gluco-oligosaccharides ; sorbitol ; glucose ; disaccharides ; immobilized enzymes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond β-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (aw) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL · mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. © 1993 John Wiley & Sons, Inc.
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  • 176
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    Biotechnology and Bioengineering 42 (1993), S. 326-332 
    ISSN: 0006-3592
    Keywords: lipolytic enzymes ; cutinases ; ester synthesis ; stability ; reversed micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani pisi recombinant cutinase, solubilized in AOT/isooctane-reversed micelles, was used to catalyze the esterification of fatty acids with aliphatic alcohols. Some relevant parameters for the enzyme activity such as pH, Wo (water/surfactant molar ratio), temperature, and substrate concentration were optimized. Maximal specific activity was obtained for hexanol. The cutinase showed selectivity for short-chain fatty acids. The stability of the microencapsulated cutinase was investigated at various concentrations of water and different values of pH. Oleic acid had a negative effect on the cutinase stability, while hexanol proved to be a strong stabilizer increasing the half-life of the enzyme about 45 times. © 1993 John Wiley & Sons, Inc.
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  • 177
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    Keywords: Candida cylindracea lipase (CCL) ; interfacial activity ; lipase purification ; polymerizable surfactant vesicles ; protein incorporation into vesicles ; triglyceride hydrolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation. © 1993 John Wiley & Sons, Inc.
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  • 178
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    Biotechnology and Bioengineering 42 (1993), S. 251-254 
    ISSN: 0006-3592
    Keywords: biosensor ; subzero temperature ; PQQGDH ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A subzero temperature operating biosensor was constructed using immobilized quinoprotein glucose dehydrogenase (PQQGDH), glassy carbon electrode, soluble electron mediator (ferrocene monocarboxylic acid), and an organic solvent, ethylene glycol, as an antifreezing reagent. Using this biosensor, glucose concentration can be determined even at -7°C. At this temperature, the response was 20% of that obtained at 20°C. This is the first study describing a subzero temperature operating biosensor. © 1993 John Wiley & Sons, Inc.
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  • 179
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    Biotechnology and Bioengineering 42 (1993), S. 260-265 
    ISSN: 0006-3592
    Keywords: fouling ; ultrafiltration ; protein aggregates ; field emission scanning electron microscopy (FESEM) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The factors contributing to protein aggregation in albumin ultrafiltration were investigated as a function of operation conditions. The nature of protein deposits was examined by electron microscopy. Protein aggregation appears to occur as a result of rapid supersaturation of protein molecules and high solvent velocity (shear) in the concentrated layer near the membrane surface. The shear occurring in the solvent flow on the membrane surface probably unfolds protein molecules and thus promotes flocculation due to collision between particles. © 1993 John Wiley & Sons, Inc.
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  • 180
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    Biotechnology and Bioengineering 42 (1993), S. 295-302 
    ISSN: 0006-3592
    Keywords: intracellular pH ; bioreactors ; cultivation ; yeast ; 9-aminoacridine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Intracellular pH (pHi) was measured on-line in a bioreactor using a fluorescent pHi indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pHi (FB-pHi) were performed. In FB-pHi cultivations, automated glucose additions were made to the culture in response to culture pHi. The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g-1 glucose in fed-batch pHi cultivations with 100 ppm glucose additions (FB-pHi-100 cultivation) vs. 0.48 g g-1 glucose in batch] and cell yield was higher (0.54 g g-1 glucose in FB-pHi-100 cultivation vs. 0.3 g g-1 glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pHi from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pHi, performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pHi cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pHi cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. © 1993 John Wiley & Sons, Inc.
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  • 181
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    Biotechnology and Bioengineering 42 (1993), S. 309-314 
    ISSN: 0006-3592
    Keywords: Papain ; immobilized papain ; enzymatic esterification ; dipeptide ester ; esterase activity ; amidase activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The esterification of some N-benzyloxycarbonyl (Z)-dipeptides in ethanol-containing water was investigated using papain as a catalyst. The esterification took place in ethanol containing a samll amount of water (2% v/v, pH 9) with free papain at room temperature. The yield (after 24 h) of the ethyl ester was in the range of 25% to 50%. Any peptide bond cleavage of the substrates was not observed during esterification, indicating that the unfavorable amidase activity of papain was well depressed under these conditions. However, dipeptides having a D-amino amino acid (Z-valyl-D-alanine) or a bulky amino acid (Z-valylvaline) at the C-terminal position could not be esterified. It was found that the immobilization of papain on Amberlite XAD-8 increased the yield of the ester significantly as compared with free papain. In the esterification of Z-valylalanine using immobilized papain, the optimum water content, pH of an added buffer, and temperature were found to be 2% (v/v), 9, and 40°C, respectively. The water content affected the yield of the product ester significantly.© 1993 John Wiley & Sons, Inc.
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  • 182
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    Biotechnology and Bioengineering 42 (1993), S. 333-338 
    ISSN: 0006-3592
    Keywords: purification fusion ; ion exchange ; membrane ; β-galactosidase ; separation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We explored the use of charged fusions for selective recovery of β-galactosidase from cell extract using a low-cost, easily scaled, fast, charge-based separation technique - ion exchange on hollow fiber ion-exchange membranes (HFIEMs). The additional charges carried by a series of anionic fusion tails allowed selective binding and release of β-galactosidase from Escherichia coli cell extract using the HFIEM cartridge. The purification factors increased with fusion length. The β-galactosidase was recovered in active form. For the longest fusion studied, more than sixfold enrichment in specific activity was attained. The specific activity of the recovered fraction is comparable with that of commercial wild-type β-galactosidase and affinity-purified fusion protein. © 1993 John Wiley & Sons, Inc.
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  • 183
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    Biotechnology and Bioengineering 42 (1993), S. 376-380 
    ISSN: 0006-3592
    Keywords: neural network ; model ; batch growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The capability of neural networks in modeling batch cell growth by providing initial conditions only is tested in this study. The neural network tested is of the back-propagation-type including a newly discovered saturation-type transfer function. The simulation and prediction results of this neural network modeling will be demonstrated. © 1993 John Wiley & Sons, Inc.
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  • 184
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    Biotechnology and Bioengineering 42 (1993), S. 381-386 
    ISSN: 0006-3592
    Keywords: alginate ; polylysine ; microcapsules ; optimization ; cut-off ; FITC-dextran ; FITC-polylysine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mammalian cells encapsulated in alginate-polylysine microcapsules are used as artificial organs in cancer research and in biotechnology. These applications require microcapsules with a reproducible mol. wt. cut-off. The high cost of the polycation, polylysine, requires an efficient preparation procedure. This article shows that the overall reported contact time of 5 minutes at ambient conditions should be increased several times in order to reach a maximal binding between the calcium alginate beads and 0.1% (w/v) polylysine solutions. An increase of the polylysine concentration from 0.0125% to 0.8% (w/v) resulted in a faster maximal binding, but the amount of polylysine bound increased also. Immersion of calcium alginate beads with a diameter of 750 μm, prepared from 1 mL alginate, in 30 mL of a 0.8% (w/v) polylysine solution, resulted in a polylysine spill of more than 89%. The time required to reach a maximal binding was related to the reaction temperature. The interaction zone between calcium alginate beads and fluorescein isothiocyanate-labeled polylysine solutions was visualized with a confocal laser scanning microscope as a function of time. Microcapsules, prepared at 40°C with 0.1% (w/v) polylysine solutions with mol. wts. between 12 and 249.2 kD, were permeable for fluorescein isothiocyanate-labeled dextran, mol. wt. 4.7, but not for 40.5 kD. Higher polylysine concentrations resulted in a membrane with a mol. wt. cut-off lower than 4.7 kD. © 1993 John Wiley & Sons, Inc.
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  • 185
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    Biotechnology and Bioengineering 42 (1993), S. 398-400 
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; bioconversion ; fructose diphosphate production ; whey ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli β-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. We showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate. © 1993 John Wiley & Sons, Inc.
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  • 186
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    Biotechnology and Bioengineering 42 (1993), S. 351-356 
    ISSN: 0006-3592
    Keywords: microencapsulation ; selection ; secretion ; yeast ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a microencapsulation selection method which allows the rapid and quantitative screening of 〉106 yeast cells for enhanced secretion of Aspergillus awamori glucoamylase. The method provides a 400-fold single-pass enrichment for high-secreting mutants, and can be straightforwardly adapted for application to growth-based selection schemes with other microorganisms and enzymes. © 1993 John Wiley & Sons, Inc.
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  • 187
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    Biotechnology and Bioengineering 42 (1993), S. 357-366 
    ISSN: 0006-3592
    Keywords: bioreactor ; insect cell culture ; high-density cell culture ; recombinant baculovirus ; chloramphenicol acetyltransferase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 μg CAT/(106 cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h. © 1993 John Wiley & Sons, Inc.
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  • 188
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    Biotechnology and Bioengineering 42 (1993), S. 394-397 
    ISSN: 0006-3592
    Keywords: cellulose ; immobilization ; fiber ; titanium oxide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fibers of a cellulose-TiO2 composite were prepared by the reaction of cellulose with titanium iso-propoxide. Enzymes were immobilized on the fibers easily and simply under mild conditions. The fibers were stable in common solvents, high ionic solutions, and over a wide range of pH values 3-10. © 1993 John Wiley & Sons, Inc.
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  • 189
    ISSN: 0006-3592
    Keywords: cellulose-binding domain ; cellulase ; cellulose ; adsorption ; affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gene fragment encoding the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi was subcloned and expressed in Escherichia coli. Transcription from the lac promoter coupled with translation from a consensus prokaryotic ribosome binding site led to the production of large quantities of CBDCex (up to 25% total soluble cell protein). The polypeptide leaked into the culture supernatant (up to 50 mg · L-1), facilitating one-step purification by affinity chromatography on cellulose. The 11-kDa polypeptide reacted with Cex antiserum. Absence of free thiols indicated that the two Cys residues of CBDCex form a disulfide bridge. It had the same N-terminal amino acid sequence as CBDCex prepared from Cex by proteolysis, plus two additional N-terminal amino acid residues (Ala and Ser) encoded by the Nhel site introduced during plasmid construction. CBDCex bound to a variety of β-1, 4-glycans with different affinities and saturation levels. Adsorption to bacterial microcrystalline cellulose was dependent on the temperature, but not on the pH. © 1993 John Wiley & Sons, Inc.
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  • 190
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    Biotechnology and Bioengineering 42 (1993), S. 421-429 
    ISSN: 0006-3592
    Keywords: bacterial contamination ; eucaryotic cultures ; detection ; chromatography ; mass spectrometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 × 103 bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 × 105 bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 × 105 and 5 × 106, respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods. © 1993 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
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  • 191
    ISSN: 0006-3592
    Keywords: penicillin G acylase ; Kluyvera citrophila ; immobilization-stabilization of penicillin G acylase ; stabilization of multimeric enzymes ; reactivation of enzyme derivatives ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)]. © 1993 John Wiley & Sons, Inc.
    Additional Material: 3 Ill.
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  • 192
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    Biotechnology and Bioengineering 42 (1993), S. 480-486 
    ISSN: 0006-3592
    Keywords: CHO ; PI-PLC ; heterologous glipiated proteins ; controlled release ; GPI anchor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. © 1993 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
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  • 193
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    Biotechnology and Bioengineering 42 (1993), S. 503-508 
    ISSN: 0006-3592
    Keywords: root culture ; fungal elicitation ; feedback inhibition ; in situ extraction ; adsorption ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The integrated recovery of solavetivone from fungus elicited “hairy root” cultures of Hyoscyamus muticus is examined using volatile organic solvents and solid-phase adsorbents in an external loop extraction configuration. Hexane and pentane are shown to be toxic when added directly to the culture; however, growth of roots is not inhibited when cultures are exposed to media saturated with these hydrocarbons. Solid-phase neutral adsorbents, XAD-7 and XAD-16, display higher capacity and better solavetivone partitioning capability than the hydrocarbons; however, their selectivity for the sesquiterpene solavetivone is poor in comparison with hexane. In both cases, the integration of product recovery through extraction resulted in a doubling of product formation by alleviating feedback repression. Implications of these results to the recovery of secondary metabolites from plant root cultures are discussed. © 1993 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
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  • 194
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    Biotechnology and Bioengineering 42 (1993), S. 509-519 
    ISSN: 0006-3592
    Keywords: Gibbs energy requirements ; chemotrophic growth ; maintenance ; anaerobic and aerobic ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A thermodynamic framework has been provided for the description of maintenance requirements of microorganisms. The central parameter is the biomass specific Gibbs energy consumption for maintenance, mE (kJ/C-mol biomass · h). A large set of data has been used including (i) a large range of different organisms (bacteria, yeasts, plant cells), (ii) mixed cultures, (iii) heterotrophic and autotrophic growth, (iv) growth under aerobic and anaerobic conditions, and (v) a large temperature range (5-75°C). It appears that only the temperature has a major influence, with an energy of activation of 69 kJ/mol. Different electron donors or electron acceptors only show a very minor influence on mE. On the basis of the data set, temperature correlations of mE have been derived for aerobic and anaerobic growth. The generalized concept for maintenance Gibbs energy is used to establish a correlation which allows the estimation of the biomass yield on electron donor as a function of C-source, electron donor, electron acceptor, N source, growth rate, and temperature. The advantage of using the mE parameter over other maintenance-related parameters (like μe, mO2, mD, γDmD) is discussed. © 1993 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
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  • 195
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    Biotechnology and Bioengineering 42 (1993), S. 548-548 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 196
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    Biotechnology and Bioengineering 42 (1993), S. 549-556 
    ISSN: 0006-3592
    Keywords: yield ; modeling ; energy balance ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two basic questions in bioprocess modeling are how many rate equations must be specified and which processes (substrate uptake, product formation, etc.) they should describe. The number of rate equations is constrained by the yield equations, which represent the balances of reducing power, energy in the form of ATP, and the various elements involved in microbial metabolism. These balances are derived from a simplified picture that divides metabolism into catabolic, anabolic, respiratory, and product formation pathways. The linear growth equation for aerobic metabolism and the Ludeking-Piret equation for product formation by fermentation are derived from these balances, and the yield coefficients are related to the metabolic parameters, YATP (P/O), etc. The use of oxygen for purposes other than respiration is included in the analysis and extends the idea of a constant “yield on available electrons” to very reduced substrates. These balances specify the number of degrees of freedom, i.e., the number of pieces of information required to complete the description of the system. This information may be in the form of measurements, knowledge of the biochemical pathways, or rate equations. The number of rate measurements available (usually two, the consumption rates of O2 and CO2) versus the number needed defines the state estimation problem in bioprocess control. Rate equations usually specify the biomass growth rate, but it may be preferable to specify the specific consumption rate of the limiting nutrient. © 1993 John Wiley & Sons, Inc.
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  • 197
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    Biotechnology and Bioengineering 42 (1993), S. 589-595 
    ISSN: 0006-3592
    Keywords: quinoline ; chemostat ; substrate inhibition ; mass balances ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The microbial degradation of quinoline by Comamonas acidovorans was studied in a laboratory scale stirred tank reactor. In continuous culture experiments using quinoline as a sole source of carbon and nitrogen, it was shown by means of mass balances that quinoline was converted completely to biomass, carbon dioxide, and ammonia. Degradation rates up to 0.7 g/L h were obtained. Measured yield coefficients Yx/s for quinoline were about 0.7 g/g, which is in agreement with the theoretical value for complete mineralization. Kinetic constants based on Haldane substrate inhibition were evaluated. The values were μmax = 0.48 h-1, Ki = 69 mg/L, and Ks 〈 1.45 mg/L. © 1993 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
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  • 198
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    Biotechnology and Bioengineering 42 (1993), S. 667-673 
    ISSN: 0006-3592
    Keywords: alcohol oxidase ; acetaldehyde ; ethanol ; continuous bioreactor ; gas phase reaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gas phase continuous production of acetaldehyde was studied with particular emphasis on the development of biocatalyst (alcohol oxidase on solid phase support materials) for a fixed bed reactor. Based on the experimental results in a batch bioreactor, the biocatalysts were prepared by immobilization of alcohol oxidase on Amberlite IRA-400, packed into a column, and the continuous acetaldehyde production in the gas phase by alcohol oxidase was performed. The effects of the reaction temperature, flow rates of gaseous stream, and ethanol vapor concentration on the performance of the continuous bioreactor were investigated. © 1993 John Wiley & Sons, Inc.
    Additional Material: 11 Ill.
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  • 199
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    Biotechnology and Bioengineering 42 (1993), S. 675-685 
    ISSN: 0006-3592
    Keywords: stringent response ; E. coli protease ; recombinant E. coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Several researchers have demonstrated that the presence of a heterologous protein in recombinant Escherichia coli elicits a response similar to the heat-shock response, which includes enhanced protease expression. The present work detects, quantifies, and characterizes intracellular protease activity in E. coli that are “shocked” by the induction of a recombinant protein, CAT, which is an endogenous protein in some E. coli strains. A novel, sodium dodecyl sulfate gelatin poly-acrylamide gel electrophoresis (SDS-GPAGE) method is used to detect, quantify, and characterize the presence of these proteases. A hypothesis is proposed which links the amplified protease activity to a temporary depletion of specific amino acid pools, and a stringent-like stress response. © 1993 John Wiley & Sons, Inc.
    Additional Material: 9 Ill.
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  • 200
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    Biotechnology and Bioengineering 42 (1993), S. 708-715 
    ISSN: 0006-3592
    Keywords: flow cytometry ; recombinant E. coli ; β-galactosidase assay ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A flow cytometric method was developed for the assay of β-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for β-galactosidase, C12FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular β-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained β-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular β-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce β-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between β-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction. © 1993 John Wiley & Sons, Inc.
    Additional Material: 9 Ill.
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