A kind of ene reductase mutant and its coding gene and application

A reductase and mutant technology, applied in the ene reductase mutant and its coding gene and application field, can solve the problems of low catalytic efficiency, low limit of ene reductase application, high production cost, etc., and achieve high reaction conversion rate and excellent Catalytic activity, no waste discharge effect

Active Publication Date: 2021-02-26
天津法莫西生物医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ene reductase has low catalytic efficiency for many α,β-unsaturated carbonyl compounds and their derivatives which are widely used in medicine and chemical industry.
For example, in the patent application number 201010216436.6, ene reductases derived from three species, Pichia guilliermondii JCM1539, Schizosaccharomyces pombe ATCC 24751, and Vanderwaltozyma polyspora NRRL Y-8283, are reported. The catalytic activity of these enzymes for many substrates is less than 1U / mg Protein, low enzyme activity limits the application of ene reductase in modern medicine, chemical industry and other fields
[0005] Therefore, it is necessary to improve the existing ene reductase to improve its catalytic activity and / or stability, thereby improving the problems of high production cost in the prior art

Method used

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  • A kind of ene reductase mutant and its coding gene and application
  • A kind of ene reductase mutant and its coding gene and application
  • A kind of ene reductase mutant and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the establishment of wild-type ene reductase gene engineering bacteria

[0034] According to the wild-type gene sequence of Thermoanaerobacter pseudothanolicus ene reductase (GenBank: ABY93685.1) collected by NCBI, the whole gene fragment was artificially synthesized after sequence optimization, and the gene was inserted into the pET22b plasmid by the gene synthesis company through NdeI and BamHI endonucleases. The ligated vector was transformed into Escherichia coli BL21 (DE3) to establish the ene reductase gene engineering bacteria.

Embodiment 2

[0035] Embodiment 2: Acquisition of ene reductase mutant gene

[0036] In this study, after obtaining the three-dimensional structure of ene reductase (3KRU / 3KRZ) from PDB, the binding simulation between the substrate of formula I (R is OH) and protein was carried out through Docking, and finally, through Pymol analysis, the possible combination of substrate and NAD was selected. Amino acids associated with binding and NAD proton transport were used as mutant amino acids.

[0037] In addition to the above-mentioned rational design, this study carried out protein engineering on ene reductase by using error-prone PCR random mutation method. In general, error-prone PCR can be performed by adjusting the reaction conditions (such as increasing the concentration of magnesium ions, adding manganese ions, changing the concentration of four kinds of dNTPs in the system or using low-fidelity DNA polymerase) when the target gene is amplified by DNA polymerase. etc.) to change the mutati...

Embodiment 3

[0053] Example 3: Small scale production of ene reductase in shake flasks

[0054] Inoculate a single microbial colony of Escherichia coli containing a plasmid encoding the target ene reductase into 100 mL of LB medium containing ampicillin (100 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH7.2). E. coli was grown in a shaker at 37°C with shaking at 250 rpm for 16 hours. The ratio of transfer was 1:100. Put 1mL of bacterial culture solution in 100mL of LB medium containing ampicillin, and shake it under the same conditions. Measure the absorbance value of the bacterial solution at 600nm regularly to monitor the growth density of the bacterial cells. When the OD600 of the culture was 0.6 to 0.8, the expression of the ene reductase gene was induced by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1 mM, and then the culture was continued overnight (at least 16 hours) . Cells were collected by centrifugation (10000 rpm, 10 min, 4°C), and ...

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Abstract

The invention discloses an ene reductase mutant, its coding gene and application. An ene reductase mutant gene, the nucleotide sequence of which is shown in SEQ ID NO.4. An ene reductase mutant gene, the nucleotide sequence of which is shown in SEQ ID NO.4. A method for preparing an ene reductase mutant, comprising the following steps: cultivating a genetically engineered bacterium of the ene reductase mutant to obtain a recombined ene reductase mutant. The invention provides an ene reductase mutant with high catalytic activity and good thermal stability, its coding gene and application.

Description

technical field [0001] The invention relates to the field of biotransformation, in particular to an ene reductase mutant, its coding gene and its application. Background technique [0002] Biotransformation technology is a technology that uses microbial cells or enzymes as catalysts to transform substances. It has the advantages of mild conditions, less side reactions, strong selectivity, low energy consumption, and environmental friendliness, and has been widely used in the fields of green chemistry and medicine. Among them, enzymes, as a common biocatalyst, play an important role in the biocatalysis process. [0003] Redox reactions are an important class of chemical reactions. The reduction of α,β-unsaturated carbonyl compounds and their derivatives to chiral compounds is a very important reaction in medicinal chemistry and organic synthesis. At present, the chiral reduction of α,β-unsaturated carbonyl compounds mainly adopts chemical methods. Reduction by chemical me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N1/21C12P17/04C12R1/19
CPCC12N9/001C12N15/70C12P17/04C12Y103/01
Inventor 马向辉宋丽
Owner 天津法莫西生物医药科技有限公司
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