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  • 1
    ISSN: 0178-515X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Different feeding strategies of galactose were employed to improve the production of anticoagulant, hirudin, by fed-batch mode of cultivation from recombinant Saccharomyces cerevisiae. The structural gene coding for hirudin was harboured with GAL10 promoter for controlled expression of hirudin and the MFα 1 signal sequence for secretion into the growth medium. A step-wise feeding of galactose was found as more suitable feeding strategy of galactose which resulted in the final hirudin volumetric productivity of 6,840 μg/l · h, than intermittent, continuous and ethanol controlled feeding of galactose. The final volumetric productivity of hirudin obtained by step-wise feeding of galactose was 3.88 fold higher compared with simple batch fermentation.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0178-515X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A medium based on less expensive nutrient sources, such as corn starch hydrolyzate (hydrol), corn steep liquor (CSL), urea and potassium phosphate was used for the growth of the yeast Phaffia rhodozyma 2A2N strain. A central composite experimental design has been employed to derive a statistical model on the effect of hydrol and CSL on carotenoid production. An initial concentration of sugars as glucose equivalent 73 g/l in hydrol and 43 g/l CSL were found optimal for the maximization of final carotenoid production in shake flask cultures. The carotenoid production was increased by adding urea and phosphate sources. Laboratory scale fermentation was performed with the optimized medium and total carotenoid production of 52.4 mg/l was obtained using constant fed-batch culture.
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  • 3
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 125 (1995), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract A sorbitol dehydrogenase was purified from the membrane fraction of Gluconobacter suboxydans KCTC 2111 (= ATCC 621) by chromatography on CM-, DEAE-, Mono S and Superose 12 columns. The purified enzyme showed a single activity band upon nondenaturing polyacrylamide gel electrophoresis (PAGE) and three subunits of 75, 50 and 14 kDa upon SDS-PAGE. When purified preparations of the enzyme were reconstituted with pyrroloquinoline quinone (PQQ), the specific enzyme activity was significantly increased (up to 9-fold). The absorption spectrum of purified sorbitol dehydrogenase in the reduced state exhibited three absorption maxima (417, 522 and 552 nm) which is in accordance with the typical absorption spectrum of cytochrome c. The 50 kDa subunit appeared as a red band on unstained SDS-gels suggesting its identity as a cytochrome. Fluorescence spectra of extracts from purified sorbitol dehydrogenase showed an excitation maximum at 370 nm and an emission maximum at 465 nm, which conformed to those of authentic PQQ. The purified enzyme showed a rather broad substrate specificity with significant activity toward D-mannitol (68%) and D-ribitol (70%) as well as D-sorbitol (100%). The PQQ-dependent sorbitol dehydrogenase described in this study is clearly different from the FAD-dependent sorbitol dehydrogenase from G. suboxydans var. α IFO 3254 strain in its cofactor requirement and substrate specificity.
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  • 5
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 [pUDF-A81]. In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentrations (OD600〉200), the extracellular LFTase accumulated to 46 000 U ml−1 (∼2.0 g l−1) which was almost 40% of total (intra- and extracellular) recombinant LFTase. The synthesized recombinant enzyme was secreted soon after gene expression was induced by IPTG. Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed-batch cultures. When lactose was added by continuous feed mode, the secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level. Therefore the induced cell culture of recombinant E. coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis. In the case of the controlled feed of lactose, the maximum activities (U ml−1) of total and extracellular LFTase were nearly 100% and 70% higher, respectively.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 5 (1989), S. 149-158 
    ISSN: 0749-503X
    Keywords: Superkiller ; double-stranded RNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The yeast chromosomal genes SK12, SK13, SK14, SK16, SK17 and SK18 repress the replication of double-stranded RNA viruses, protecting the host from the otherwise lethal effects of the virus. We cloned and sequenced the SK13 gene and found that it encodes a 163 kDa protein including a typical nuclear localization signal. Cell fractionation experiments show that the SK13 gene product is indeed tightly associated with nuclei and that the putative nuclear localization sequence directs β-galactosidase into the nucleus. However, fusion of a part of the SK13 protein lacking this signal with β-galactosidase directs β-galactosidase into the nucleus, suggesting the presence of a second nuclear localization signal. The SK13 gene is only essential in the presence of an M double-stranded RNA virus.
    Additional Material: 4 Ill.
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  • 7
    ISSN: 0749-503X
    Keywords: yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
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  • 8
    ISSN: 0749-503X
    Keywords: α1-antitrypsin ; Saccharomyces cerevisiae ; Hansenula polymorpha ; Pichia pastoris ; glycosylation ; secretion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Human α1-antitrypsin (α1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human α1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant α1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant α1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted α1-AT. The human α1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of α1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of α1-AT in S. cerevisiae. The α1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of α1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
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  • 9
    ISSN: 1573-6776
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The enzymatic conversion of ethanol into acetaldehyde using dried whole cells ofHansenula polymorpha was tried in a gas-solid bioreactor. The bioreactor could be maintained stably over one month at 35°C with complete conversion in the water content under 8% without any serious problems.
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  • 10
    ISSN: 1573-6776
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The enriched medium based on yeast nitrogen basc(YNB)increased hirudin synthesis and secretion in rccombinant Saccharomyces cerevisiae in batch and fed-batch cultures. Fed-batch fermentation with the defined medium yielded 342mg hirudin/l but supplementation of yeast extract increased the final hirudin concentration to 461mg hirudin/l. The defined medium, however, produced the product protein with higher purity of 21% and hence will allow easy separation of secreted hirudin from other contaminated polypeptides present in the growth medium. In a continuous culuture, the defined medium yielded higher concentrations of cell mass and hirudin than the complex medium.
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