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  • 1
    Electronic Resource
    Electronic Resource
    Investigating expression systems for the stable large-scale production of recombinant L-leucine-dehydrogenase from Bacillus cereus in Escherichia coli (2000)
    Springer
    Applied microbiology and biotechnology 53 (2000), S. 668-673 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The established Escherichia coli expression vectors ptrc99a, pKK223-3, pPLλ, pAsk75, pRA95, and pRA96, which differ in copy number, mode of induction, selection marker, and use of par sequences for stabilization, were investigated for the stable expression of recombinant L-leucine dehydrogenase from Bacillus cereus with a view to large-scale production. Best results were achieved with pIET98, a runaway-replication system derived from pRA96. Expression of L-leucine dehydrogenase was controlled by its constitutive B. cereus promoter and depended on host strain, cultivation temperature, induction time, and media composition. After cell cultivation at 30 °C and shifting to 41 °C to induce plasmid replication, E. coli BL21[pIET98] yielded 200 U LeuDH/mg protein, which corresponds to 〉50% of the soluble cell protein. Continuous cultivation in a semisynthetic high-cell-density medium verified structural and segregational stability over 100 generations in the absence of a selection pressure.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002539900290
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system (2000)
    Springer
    Applied microbiology and biotechnology 54 (2000), S. 361-369 
    Details
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg l−1 of the fusion protein (420 mg l−1 of the recombinant hCT precursor) within 14 h, reaching 45 g l−1 cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor l−1 h−1) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-l scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g−1 yeast extract).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002530000406
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems (2000)
    Springer
    Bioseparation 9 (2000), S. 269-276 
    Details
    ISSN: 1573-8272
    Keywords: Agrimul NRE 1205 ; aqueous two-phase systems ; cloud point extraction ; tagged cutinase ; tag design for protein extractions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point. In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example. Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag. In contrast, the extraction yield of wild type cutinase was 2–3% only. The detergent concentration and the temperature are the main parameters to optimize the extraction yield. Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1011190713820
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    Paper (German National Licenses)
    Fulltext
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