A kind of method applying perakine reductase to synthesize chiral alcohol

A technology of reductase and chiral alcohol, which is applied in fermentation and other fields, can solve the problems that chiral alcohol cannot be widely used, and achieve the effects of easy preparation, good stability and mild reaction conditions

Active Publication Date: 2020-05-08
ZHEJIANG UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

At present, the enzymes that can be applied to asymmetric reduction of ketones mainly include short-chain dehydrogenases and ketoreductases, but due to the limitations of stability, substrate spectrum, catalytic efficiency and enzyme production, they cannot be widely used in hand. The synthesis of chiral alcohols, so researchers are still looking for new ketoreductases that can be used for the synthesis of chiral alcohols

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  • A kind of method applying perakine reductase to synthesize chiral alcohol
  • A kind of method applying perakine reductase to synthesize chiral alcohol
  • A kind of method applying perakine reductase to synthesize chiral alcohol

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Embodiment 1

[0040] The expression of embodiment 1Perakine reductase

[0041] The Perakine reductase expression vector was constructed in previous research (Sun L, Ruppert M, Sheludko Y, Warzecha H, Zhao Y, J. Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaline network of the plantRauvolfia. Plant Mol Biol. 2008, 67(5): 455-67.), heat shock conversion to E.coli M15 competent cells were used to obtain engineering bacteria expressing Perakine reductase. The engineering bacteria expressing Perakine reductase were inoculated into the LB medium containing 50 μg / ml ampicillin and 25 μg / ml kanamycin and cultured with shaking at 37°C for 12 hours. Transfer to 1L LB medium containing the same concentration of ampicillin and kanamycin, when the optical density of the culture medium is OD 600 When it reaches 0.6, add isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.3m...

Embodiment 2

[0042] The expression of embodiment 2 glucose dehydrogenase

[0043] The glucose dehydrogenase gene (GenBank: D10626.1) encoding Bacillus megaterium IAM1030 is shown in the sequence listing GDH-DNA, and the amino acid sequence is shown in the sequence listing GDH-AA. The glucose dehydrogenase gene was fully synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. and then connected to the pET-28a(+) vector. After sequencing and verifying the sequence, it was transformed into E.coli BL21(DE3) competent cells by heat shock to obtain glucose Dehydrogenase expression engineering bacteria. The glucose dehydrogenase-expressing engineered bacteria were inoculated into LB medium containing 50 μg / ml kanamycin and cultured with shaking at 37°C for 12 hours. Transfer to 1L LB medium containing the same concentration of kanamycin, when the optical density of the culture medium OD 600 When it reaches 0.6, add IPTG with a final concentration of 0.1mM to induce at 20°C for 16h, centrifuge...

Embodiment 3

[0044] Embodiment 3Perakine reductase and the purification of glucose dehydrogenase

[0045] Perakine reductase-expressing engineered bacteria or glucose dehydrogenase-expressed engineered bacteria cells were resuspended in 20ml lysate (10mM imidazole, 50mM NaH 2 PO 4 , 300mM NaCl, pH 8.0), shake well and add 1-2mg / ml lysozyme, after ice bath for 40min, put ultrasonic breaker 3 times, 3min each time, each interval of 15min. The broken liquid was centrifuged at 22000×g for 50 min, and the obtained supernatant was the crude enzyme liquid. Use Ni-NTA as the purification material, the column volume is 3ml, 15ml lysate equilibrates the Ni-NTA column, loads the sample crude enzyme solution at a rate of 1ml / min, rinses with buffer (20mM imidazole, 50mM NaH 2 PO 4 , 300mM NaCl, pH8.0) to remove unadsorbed protein, and finally eluted with elution buffer (250mM imidazole, 50mM NaH 2 PO 4 , 300mM NaCl, pH8.0) to collect the target protein by elution, and use 5L Kpi buffer (50mM KH ...

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Abstract

The invention discloses a method for synthesizing chiral alcohol using a Perakine reductase. The method is characterized in that different prochiral ketones are taken as substrates, and the chiral alcohol is reduced and synthesized through a Perakine reductase catalytic system, wherein the catalytic system is composed of the Perakine reductase and a coenzyme regeneration system. The method has theadvantages that the reaction steps are simple, the reaction conditions are mild, the method is environmentally friendly, the catalytic efficiency is high, the enzyme purification is simple, the stability is good, three-dimensional selectivity is high, and the method has better industrial application developing prospects in the industrial synthesis fields of the chiral alcohol and related chiral medicines.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and specifically relates to a method for asymmetrically reducing latent chiral ketones using Perakine reductase as a catalyst to obtain chiral alcohols of optical alcohols. Background technique [0002] Chiral alcohols are a class of extremely important intermediates in the synthesis of fine chemicals and chiral drugs, and their synthesis has always attracted the attention of researchers. At present, chemical and biological catalysts are mainly used to catalyze prochiral ketones to obtain chiral alcohols by means of asymmetric hydrogen transfer (ATH) and asymmetric hydrogenation (AH). Compared with chemical catalysis, biocatalysis has the characteristics of high efficiency and environmental protection, which is more in line with the current development trend of "green chemistry". Biocatalysis methods mainly include whole-cell catalysis, crude enzyme solution catalysis and pure enzym...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/02
CPCC12P7/02
Inventor 孙莲莉陈媛媛邵娜娜周韵
Owner ZHEJIANG UNIV
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