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Recombinant bacterium of lipase expressing R configuration selectivity and application thereof

A selective and recombinant bacteria technology, applied in the direction of enzymes, bacteria, hydrolytic enzymes, etc., can solve the problems of different stereoselective catalytic functions, complex processes, high costs, etc.

Active Publication Date: 2018-09-07
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although lipases are widely distributed in the microbial world, their stereoselective catalytic functions are different. A single enzyme preparation is obtained by separating and purifying microbial fermentation products to carry out catalytic reactions, often requiring more complicated processes and higher costs. , it is not easy to be adopted in production

Method used

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  • Recombinant bacterium of lipase expressing R configuration selectivity and application thereof
  • Recombinant bacterium of lipase expressing R configuration selectivity and application thereof
  • Recombinant bacterium of lipase expressing R configuration selectivity and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Discovery of R-configuration-selective lipase AflB

[0031] Through the mining of the protein database, an Aspergillus fumigatus Af293lipase (GenBank: XP_749106) amino acid sequence was found, and it was found that the enzyme showed a low sequence identity with CalB, a lipase widely used in industry. Identity=31%, and contained a CalB that did not have The N-terminal alpha-helical domain. The N-terminal α-helical domain is favorable for the binding of R-configuration naproxen ((-)-2-(6-methoxy-2-naphthyl)propionic acid) through homology modeling and substrate molecular docking . And on the basis of the molecular docking, the corresponding amino acid residues in the substrate pocket were replaced by computer simulation, and Aspergillus fumigatus Af293lipase was further modified to make it more inclined to bind R-configuration naproxen. Finally, the modified Aspergillus fumigatus Af293lipase was named R-configuration-selective lipase AflB, which consisted of 438 amino a...

Embodiment 2

[0034] Expression and Purification of R Configuration Selective Lipase AflB

[0035] 1. Construction of recombinant plasmids

[0036] The lipase AflB coding gene sequence is shown in SEQ ID NO.2. The lipase AflB gene was obtained through total gene synthesis, and then cloned into the pET28a plasmid to obtain the plasmid pET28a-AflB.

[0037] 2. Obtaining of lipase AflB producing bacteria with recombinant R configuration selectivity

[0038] Take a tube of Escherichia coli SHuffle T7Competent E.coli (Cat.C3026, NEB) competent cells, thaw on ice, transfer 50ul to a pre-cooled 1.5ml centrifuge tube with a pre-cooled pipette tip, and then add the recombinant Plasmid pET28a-AflB 1ul, shake gently to mix, place in ice bath for 30 minutes. Quickly transfer to a 42°C water bath for exactly 40 seconds. Quickly transfer to ice and chill for 2 minutes. Add 500ul LB culture medium to each tube and incubate at 37°C for 45 minutes. Spread 20ul on a plate containing antibiotics (50mg / m ka...

Embodiment 3

[0058] Determination of Enzymatic Properties of Recombinant R Conformation Selective Lipase AflB

[0059] 1. Optimum reaction pH value of lipase AflB with recombinant R configuration selectivity

[0060] At 40°C, with tributyrin as substrate, pH 3.0, 4.0 and 5.0 (50mM sodium acetate buffer), pH 6.0 and pH 7.0 (50mM phosphate buffer), pH 8.0 (50mM Tris -HCl buffer), pH 9.0 and pH10.0 (50mM glycine buffer) conditions, measure the relative enzyme activity ( figure 2 ).

[0061] 2. Optimum reaction temperature of lipase AflB with recombinant R configuration selectivity

[0062] Using tributyrin as a substrate, at pH 7.0 (50mM phosphate buffer), at 20°C, 30°C, 40°C, 45°C, 50°C, 60°C, 70°C and 80°C, respectively Carry out catalytic reaction under the condition, measure the relative enzymatic activity ( image 3 ).

[0063] 3. Selectivity of recombinant R-configuration-selective lipase AflB to substrates with different chain lengths

[0064] Triacetin (TC2), tributyrin (TC4), ...

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Abstract

The invention discloses a recombinant bacterium of lipase expressing R configuration selectivity and application thereof. A lipase gene with R configuration selectivity is inserted into a multiple cloning site of a pET28a plasmid to obtain a recombinant plasmid; the recombinant plasmid is transformed into colibacillus SHuffle T7 to obtain the recombinant bacterium; the gene sequence of the lipasegene with the R configuration selectivity is a gene sequence of the following 1) or 2), wherein 1) is a DNA sequence shown as SEQ ID NO.2 in a sequence table; 2) is a DNA sequence with 90% homology orabove with the DNA sequence defined in 1) and used for encoding protein with esterolysis enzymatic activity. The recombinant lipase has higher R-configuration steric selectivity and catalytic activity, more than 85% of the enzyme activity is reserved under a condition with higher temperature, and the recombinant lipase can be used for splitting racemization 2-aryl-methyl propionate substances, and obtain optical pure S-type 2-aryl-methyl propionate substances.

Description

technical field [0001] The present invention relates to an R-configuration-selective lipase AflB and its encoding gene, as well as a carrier containing the encoding gene, an engineering bacterium and its application in chiral resolution of racemic 2-aryl propionic acid substances . Background technique [0002] Lipase widely exists in animals, plants and microorganisms. It is a kind of enzyme that catalyzes hydrolysis or forms ester bonds. The substrates are usually esters with fatty acid chains larger than ten carbon atoms. Lipase can catalyze various chemical reactions such as hydrolysis, esterification, and transesterification, and is a very important industrial catalyst. In addition, the broad-spectrum / special specificity of lipase to substrate endows lipase with great application value in the synthesis of ester bond compounds and chiral drug synthesis and other industries. [0003] Due to the increasing demand for chiral drugs and intermediates, the use of biological ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P41/00C12P7/62C12R1/19
CPCC12N9/20C12P7/62C12P41/001C12Y301/01003
Inventor 王永华蓝东明袁红黄伟谦杨博
Owner SOUTH CHINA UNIV OF TECH
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