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Fermentation And Immobilization Of Recombinant E. Coli Expressing Nitrilase For Biosynthesis Of (R)-(-)-mandelic Acid From Mandelonitrile

Posted on:2012-08-30Degree:MasterType:Thesis
Country:ChinaCandidate:J F LiuFull Text:PDF
GTID:2121330332975760Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
Nitrilases are widely used in organic synthesis and environmental protection. At present, the market of optically pure products catalyzed by nitrilases is extended increasingly. For example, mandelic acid, as the product from mandelonitrile, is an important building block in pharmaceutical industry and chemical synthesis. The fermentation of recombinant E. coli for production of nitrilase and the immobilization of whole cells for biosynthesis of (R)-(-)-mandelic acid from mandelonitrile were investigated in this paper.Firstly, the production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters including medium composition, inducer, induction condition, pH and temperature were systematically examined. For the batch culture, the highest nitrilase production with good enantioselectivity (yielding (R)-(-)-mandelic acid with >95% enantiomeric excess) was obtained when the the fermentation was carried out at 37℃, initial pH 7.0 without control and the E. coli was induced with 0.2 mM IPTG at 4.0 h. Furthermore, the nitrilase production could be significantly enhanced by adopting a glycerol feeding strategy. The enzyme expression was also authenticated by the sodium dodecyl phosphate-polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, the growth of the organism, specific activity and volume production of recombinant nitrilase were increased by 9.0-,5.5- and 50-folds, respectively.Secondly, the recombinant E.coli cells expressing nitrilase were entrapped in alginate gel and the main parameters such as the concentrations of cell and alginate, the time of cross-linking treatment with polyethyleneimine (PEI) and the methods for the storage of immobilized cells were optimized. The substrate tolerance of the cells was improved after immobilization. The half-life (194 min) of the immobilized cells treated with PEI and glutaraldehyde (GA) at 60℃is 6-folds and 2.5-folds longer than free cells and immobilized cells without treatment with PEI or GA respectively. The conversion of mandelonitrile still remained 39.8% after 19 batches of reaction and the specific production of immobilized biocatalyst (2.37 g mandelic acid/g wet cell weight) was improved by 1.9-folds as compared to free cells. Finally,1.62 g (R)-(-)-mandelic acid with enantiomeric excess of 98.2% was prepared by employing 3.0 g (wcw) of immobilized cells and 3.15 g cross-linking immobilized cells with a yield of 60.8%.
Keywords/Search Tags:Nitrilase, Optimization, Fermentation, Recombinant Escherichia coli, Fed-batch culture, Immobilization, Mandelonitrile, (R)-(-)-Mandelic acid
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