Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application

A technology of short-chain dehydrogenase and genetically engineered bacteria, applied in short-chain dehydrogenase and application fields

Inactive Publication Date: 2012-12-19
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reductive conversion of COBE with the cell-free extract and cell acetone dry powder prepared by it obtained an ideal result of 99% e.e., but the molar conversion rate was only 64%

Method used

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  • Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application
  • Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application
  • Short-chain dehydrogenase CPE (Cytopathic Effect) gene, coding enzyme, carrier, recombination engineering bacteria and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 short-chain dehydrogenase CPE gene, gene coding enzyme, carrier, recombinant genetically engineered bacterium

[0047] 1. Materials

[0048] (1) Strains and plasmids

[0049] Strains: Candida parapsilosis (Candida parapsilosis CDC317) was purchased from the American Type Culture Collection (ATCC), No. MYA-4646), plasmid pET21d was derived from Novagen (Madison, WI, USA), E.coli DH5α, E.coli BL21 (DE3) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0050] (2) Reagents

[0051] pMD-19T kit, Taq enzyme, dNTP mixture (single nucleotide mixture), IPTG, high-ligation (high-efficiency ligase), restriction endonuclease purchased from TAKARA company, 4-chloroacetoacetate ethyl ester (COBE) Purchased from Alfa Aesar, Yeast Genomic DNA Extraction Kit, Agarose Gel DNA Recovery Kit, Common Plasmid Mini-Extraction Kit, Common DNA Product Purification Kit were purchased from TIANGEN Company, Lowry Method Protein Quantitative Analysis...

Embodiment 2

[0111] The enzymatic property determination of embodiment 2 short-chain dehydrogenase CPE

[0112] 1. Determination of optimal temperature enzyme activity

[0113] Take a 1.5ml centrifuge tube, add 100 μL DMSO, 5 μL COBE, 380 μL PBS (pH=7.0), and incubate at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C for 10 minutes , then add 5 μL of PBS solution of 10 mg / ml NADPH (pH 7.0) and 10 μL of the supernatant prepared by the method of step (3) of Example 1 (obtained by centrifuging 100 ml of the fermented culture broth with a wet cell concentration of 4 mg / mL), at 340 nm The absorbance value was measured at the place, and the kinetic curve of enzyme activity was made. The results are shown in Figure 4 shown.

[0114] Test results( Figure 4 (shown in curve a) shows that the optimum temperature for short-chain dehydrogenase CPE is 40°C, and the enzyme activity decreases rapidly when it is below 35°C or above 40°C.

[0115] 2. Determination of optimal pH enzyme activity

[011...

Embodiment 3

[0125] Example 3 Short-chain dehydrogenase CPE reduces COBE

[0126] The COBE reduction reaction system is as follows:

[0127]

[0128] Glucose dehydrogenase 75U / L, recombinant glucose dehydrogenase derived from Bacillus subtilis according to literature reports (Zhou Liping, Zhao Yan, Wang Huifang, Ding Jianxia, ​​Cloning and expression of Bacillus subtilis glucose dehydrogenase, Journal of Jiangsu University (Medical Edition) ), 2004, 14(1), 7-10.)

[0129] The supernatant was obtained by centrifuging 5 ml of the fermented culture broth with a wet cell concentration of 4 mg / mL.

[0130] Mix the reduction reaction system and react overnight at 30°C with a 180rpm shaker. After the reaction, extract the reaction solution twice with 3mL ethyl acetate, take the organic layer and wash it with anhydrous Mg 2 SO 4 Dehydrate and filter to obtain about 2.5ml of filtrate, which is a mixed solution containing S-4-chloro-3-hydroxybutyrate ethyl ester (S-CHBE) product. The filtrate ...

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Abstract

The invention discloses a short-chain dehydrogenase CPE (Cytopathic Effect) gene, a gene coding enzyme, a carrier, a recombination engineering bacteria and an application of the engine coding enzyme in an asymmetric reductive carbonylation compound. The short-chain dehydrogenase CPE has relatively high conversion rate and e.e. value; and the reductase with relatively low Km value can keep enzyme activity for a longer time under a condition that the concentration of substrate is relatively low; and the quantity of added enzyme can be reduced in the actual industrial production; and the cost can also be reduced; and moreover, the maximum reaction speed can be reached when the substrate with low concentration is added, so that the production period of unit weight products can be reduced in the actual industrial production, and the energy consumption and the labor cost can be decreased.

Description

(1) Technical field [0001] The invention relates to a short-chain dehydrogenase and its application, in particular to the short-chain dehydrogenase CPE gene, coding enzyme, carrier, recombinant engineering bacteria and its application in asymmetric reduction of carbonyl compounds. (2) Background technology [0002] β-hydroxy ester is an important part of pharmaceuticals, pesticides, materials and other fine chemical raw materials, such as 4-chloro-3-hydroxybutyric acid ethyl ester (CHBE), which is an important organic synthesis intermediate, and there are three in the molecule Functional groups (-OH, -Cl, -COOH) can be introduced into a variety of drug intermediates through chlorine displacement reactions, reduction reactions, etc. Its chiral monomer (R / S)-CHBE is a very promising chiral substance. R-CHBE can be used as a key intermediate in the synthesis of L-carnitine, (-)-large lactam A, R-γ-amino-β-hydroxybutyric acid, and can also be converted into negative mycin. (S)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/15C12N1/19C12N1/21C12P7/62
Inventor 殷晓浦谢恬王秋岩谌容裴晓林曹丹赵淑娟
Owner HANGZHOU NORMAL UNIVERSITY
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