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Method for preparing (S)-styrene glycol with carbonyl reduction enzyme and pyrimidine nucleoside acid transhydrogenase couplet

A phenylethylene glycol, coupling technology, applied in the field of preparing (S)-phenylethylene glycol by coupling carbonyl reductase and pyrimidine nucleotide transhydrogenase, which can solve the problem of high price, unstable coenzyme, etc. question

Active Publication Date: 2009-02-18
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Coenzymes are generally unstable and expensive
In the asymmetric reduction reaction catalyzed by oxidoreductase, the inability to recycle the coenzyme is the "bottleneck" factor that limits its industrial application

Method used

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  • Method for preparing (S)-styrene glycol with carbonyl reduction enzyme and pyrimidine nucleoside acid transhydrogenase couplet

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 The cultivation of Candida parapsilosis and Escherichia coli:

[0052] Candida parapsilosis medium composition: glucose 4%, yeast extract 0.5%, (NH4) 2 HPO 4 1.3%, KH 2 PO 4 0.7%, ZnSO 4 .7H 2 O 0.03%, NaCl 0.01%, pH 7.0. The strains were inoculated into 250 mL shake flasks with 50 mL of culture medium, and cultured with shaking at 30°C and 150 rpm for 48 hours.

[0053] Escherichia coli LB medium composition: peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0. Ampicillin (100 μg / mL) and chloramphenicol (34 μg / mL) were added during cultivation. Add 1.5% agar powder to the solid medium. The strains were inoculated into a 250 mL shake flask containing 50 mL of LB liquid medium, and cultured at 37° C. with shaking at 200 rpm for about 12 hours.

Embodiment 2

[0054] Example 2 Obtaining of Candida parapsilosis genome and Escherichia coli genome:

[0055] After the bacterial cell culture, the bacterial cell was centrifuged at 6,000 rpm for 20 min, washed twice with saline, and the cells were collected, and the genome was extracted using a genomic DNA extraction kit Genomic DNA Extraction Miniprep System (VIOGENE Company).

Embodiment 3

[0056] Example 3 Obtaining of rcr gene:

[0057] Using the primer RCR_F containing the NdeI restriction site and the primer RCR_R containing the XhoI restriction site,

[0058] RCR_F: 5'-ATCGATCG CATATG TCAATTCCAT CAAGCCAGTAC-3'(NdeI), RCR_R: 5'-TGACT CTCGAG TGGATTAAAAACAA CTCTACCTTC-3' (XhoI);

[0059] PCR reaction system: ddH 2O 37μL, 10×Reaction Buffer 5μL, 25mmol / L dNTP 0.5μL, 50pmol / μL primers RCR_F and RCR_R 1μL each, Candida parapsilosis genome 5μL, 5U / μL Taq DNA polymerase 0.5μL; PCR reaction conditions: 94℃ Pre-denaturation for 5 min; 30 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min; 72°C for 10 min; PCR reaction to obtain the rcr gene fragment.

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Abstract

A method for preparing (S)-phenylethylene glycol by coupling of carbonyl reductase and pyrimidine nucleotide transhydrogenase, which belongs to the technical field of biocatalysis asymmetric transformation. In the invention, (R)-carbonyl reductase, (S)-carbonyl reductase and pyrimidine nucleotide transhydrogenaseA, B are respectively constructed to two co-expression vectors, and the constructed recombinant plasmid pETDuet-rcr-scr and pACYCDuet-pnta-pntb are co-transformed into Escherichia coli to obtain recombinant strain E. Coli BL21(pETDuet-rcr-scr / pACYCDuet-pnta-pntb). The accession number is CCTCC NO: M208126. The recombinant strain not only realizes one-step transformation from substrate (R)-phenylethylene glycol to product (S)-phenylethylene glycol, but also removes a restriction of coenzyme regeneration cycle in a transformation reaction. The method provides an effective way for producing chiral alcohol through catalytic reaction of enzyme and regeneration of in-situ coenzyme under high substrate concentration.

Description

technical field [0001] Utilize (R), (S)-carbonyl reductase and pyrimidine nucleotide transhydrogenase A, the method for the coupling system of four enzyme coexpression of B four enzymes to prepare (S)-phenylethylene glycol, the present invention relates to utilizing gene Engineering technology constructs (R), (S)-carbonyl reductase genes and A, B pyrimidine nucleotide transhydrogenase genes on two compatible co-expression vectors, and simultaneously transfers the four genes into The method for efficiently preparing (S)-phenylethylene glycol by using a constructed recombinant strain in Escherichia coli cells and its application belong to the technical field of biocatalytic asymmetric transformation. Background technique [0002] Optically pure (S)-phenylethylene glycol is an indispensable and important chiral additive in liquid crystal materials, and is also an important intermediate for the preparation of optically active medicines, pesticides and functional materials. Asym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/53C12N15/70C12P7/22C12R1/19
Inventor 张荣珍徐岩
Owner JIANGNAN UNIV
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