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Study on Asymmetric Biosynthesis of D-pseudoephedrine by Recombinant Bacillus Subtilis

Author: PengYanHong
Tutor: ShiGuiYang
School: Jiangnan University
Course: Biochemical Engineering
Keywords: Bacillus subtilis Carbonyl reductase Integrate Cre-loxP system Biotransformation
CLC: TQ463
Type: Master's thesis
Year: 2011
Downloads: 63
Quote: 1
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Abstract


Ephedrine (Ephedrine), due to having a larger pharmacological effects, and are widely used in clinical medicine. Biotransformation prepared optically pure ephedrine mild reaction conditions, high stereoselectivity, one of the ideal way of the production of ephedrine, has a large potential for development; the biotransformation process needs the coenzyme to provide reducing power . The papers focus on the problem the biotransformation processes encountered early in our laboratory coenzyme regeneration, Bacillus subtilis (Bacillus subtilis) containing glucose dehydrogenase GDH for host its own building carbonyl reductase gene mldh free expression strain and integration The expression strain, in order to achieve expression of mldh in Bacillus subtilis and complete the regeneration of the coenzyme by the GDH intracellular. The B. subtilis Wb600 as a host, Bacillus subtilis and the promoter of the gene PrpsD rpsD and terminator TrpsD the expression elements the mldh of carbonyl reductase gene, together with expression elements connected together to obtain plasmid pHY300plk-PrpsD-the carrier pHY300plk on Bacillus subtilis mldh-TrpsD; to get free expression strain B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD) further the recombinant plasmid was transformed into B. subtilis Wb600. This episomal expression strains whole cell biotransformation reactions was found, in the case of the presence of glucose, which can be transformed into the substrate 1 - phenyl - 2 - methylamino acetone (MAK) to produce d-pseudoephedrine; 0.2 g culture time free expression strain wet cells 12 h, 0.4 mg of MAK and 18 mg of glucose conversion reaction, the yield up to 97.5 mg / L, substrate molar yield of 24.1%. Amylase gene on the chromosome of Bacillus subtilis after amyE integration sites kan as a resistance marker gene on pUB110; Construction integrative expression vector pUCl9-5E-PMT-loxpkan-3E of kan containing the loxP sites at both ends; the strains obtained in the B. subtilis Wb600 (5E-pmt-loxpkan-3E) vector was transformed into B. subtilis Wb600. As a template the plasmid pSH47 amplified CRE recombinase gene Cre, the Bacillus subtilis PxylR gene promoter, construct of expression plasmid pHY300PLK-PxylR-cre-trpsD; This vector was transformed into B. subtilis Wb600 (5E-pmt-loxpkan- 3E), xylose induction cre gene expression, knockout resistance gene kan; the longer final integrated expression strains the B. subtilis Wb600 (5E-pmt-loxp-3E by the subculture the the lost plasmid pHY300plk-PxylR-cre-trpsD,) . The integrated recombinant strain B. subtilis Wb600 (5E-pmt-loxp-3E) experimental whole cell conversion reaction, results of incubation time 0.2 g wet cells for 12 h integrated recombinant strain, 0.3 mg of MAK and 6 mg of glucose into the reaction, d-pseudoephedrine production of 47.2 mg / L, the substrate molar conversion rate of 15.5%. This study has successfully mldh exogenous gene integrated into the chromosome of Bacillus subtilis, to achieve the stable expression of the target gene in the host bacteria, and the completion of the regeneration of the coenzyme. The results for the use of recombinant B. subtilis biotransformation production of d-pseudoephedrine useful exploration.

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CLC: > Industrial Technology > Chemical Industry > Pharmaceutical chemical industry > Production of organic compounds in drug
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