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Lipase mutant and application thereof

A lipase and mutant technology, applied in application, enzyme, hydrolase and other directions, can solve the problems of poor solubility, low target protein expression, weak catalytic activity, etc., to reduce the amount of enzyme, reduce the reaction volume, and improve the catalytic activity. active effect

Active Publication Date: 2021-02-19
JINLIN ASYMCHEM PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In industrial production, most wild-type lipases have low catalytic efficiency, poor stereoselectivity, and weak stability. When they are expressed in common engineering bacteria by genetic engineering methods, they have low target protein expression, poor solubility, and catalytic activity. Weakness and other disadvantages, so there are not many lipases that can be widely used

Method used

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  • Lipase mutant and application thereof
  • Lipase mutant and application thereof
  • Lipase mutant and application thereof

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Experimental program
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Embodiment approach

[0034] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecules in the above recombinant plasmids are placed in appropriate positions of the recombinant plasmids, so that the above DNA molecules can be replicated, transcribed or expressed correctly and smoothly.

[0035] Although the qualifier used in the present invention is "contains" when limiting the above-mentioned DNA molecule, it does not mean that other sequences irrelevant to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cutting sites at both ends of the DNA sequence, or additionally add start codons, stop codons, etc., therefore, if using A closed-ended statement will not truly cover these situations.

[0036] The ...

Embodiment 1

[0048] Take 20 mg of substrate 1 / substrate 2 / substrate 3 respectively, add 2 mg of resuspended lipase or its mutant sludge, 0.1 M pH 8.5 Tris-Cl Buffer to the reaction system to supplement 500 μL of the system, 200 rpm, constant temperature reaction at 30 °C for 16 h. Add 2 times the volume (1 mL) of acetonitrile to the system, make it fully uniform, and centrifuge at 12,000 rpm for 3 minutes to observe whether there is stratification. If there is stratification, it is necessary to add acetonitrile:purified water = 1:1 mixture until no Separate the layers, and finally take 200 μL of centrifuged supernatant into 400 μL of acetonitrile: purified water = 1:1 mixture, mix well and send a sample to HPLC to detect the conversion rate. The response characteristics of some mutants are shown in Table 1 (with the solubility of protein expression):

[0049] Table 1

[0050]

[0051]

[0052] Compared with the mother parent (-), the multiple of activity increase is indicated by +,...

Embodiment 2

[0055] Take 20 mg of substrate 1 / substrate 4 respectively, add 2 mg of resuspended lipase or its mutant sludge, 0.1 M pH 8.5 Tris-Cl Buffer to the reaction system to supplement 500 μL of the system, 200 rpm, The reaction was carried out at a constant temperature of 30 °C for 16 h. Add 2 times the volume (1 mL) of acetonitrile to the system, make it fully uniform, and centrifuge at 12,000 rpm for 3 minutes to observe whether there is stratification. If there is stratification, it is necessary to add acetonitrile:purified water = 1:1 mixture until no Separate the layers, and finally take 200 μL of centrifuged supernatant into 400 μL of acetonitrile: purified water = 1:1 mixture, mix well and send a sample to HPLC to detect the conversion rate. The response characteristics of some mutants are shown in Table 2:

[0056] Table 2

[0057]

[0058]

[0059]

[0060] Compared with the parent (-), the multiple of the activity increase is indicated by +, + indicates an increa...

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Abstract

The invention discloses a lipase mutant and an application thereof. According to the lipase mutant and the application thereof, wherein the lipase mutant has a sequence with amino acid mutation of a sequence shown in SEQ ID NO: 1, and sites with amino acid mutation comprise a V154L site. The lipase mutant provided by the invention realizes the change of protein structure and function, the solubility of the lipase mutant expressed in escherichia coli is obviously improved, and the catalytic activity of enzyme is greatly improved. And in practical application, the enzyme dosage is greatly reduced, the reaction volume is reduced, the post-treatment difficulty is reduced, and the lipase mutant is suitable for industrial production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lipase mutant and its application. Background technique [0002] Many chiral acids and alcohols (such as α-substituted propionic acid compounds) are active and are important chiral units in the synthesis of chiral drugs. The separation of chiral drugs of acids, alcohols and esters is generally to first chemically synthesize the corresponding racemates of methyl ester, ethyl ester or propyl ester, etc., and then use lipase or esterase to perform stereoselective hydrolysis to obtain a single pair Chiral units in enantiomeric configuration. For example, the herbicide (R)-α-phenoxypropyl ester and the anti-inflammatory drug (S)-phenylpropanol are all converted into single active isomers by stereoselective lipase. [0003] Lipase has high stereoselectivity, can catalyze chiral resolution, and prepare organic synthesis intermediates such as single chiral alcohols, amines and esters (re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12P7/40
CPCC12N9/20C12Y301/01003C12P7/40Y02E50/10C12N15/70C12P41/005C12P9/00C12P7/62
Inventor 洪浩詹姆斯·盖吉肖毅张娜焦学成贾如刘文敬刘冶
Owner JINLIN ASYMCHEM PHARM CO LTD
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