A lipase mutant with improved catalytic activity and its application

A technology for improving catalytic activity and lipase, which is applied in the field of enzyme engineering and can solve problems such as the inability to meet industrial needs.

Active Publication Date: 2021-01-12
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with lipases from other microbial sources, Thermomyces lanuginosus lipase has better thermal stability, but it still cannot meet the industrial needs; in addition, the catalytic activity of Thermomyces lanuginosus lipase is still Affected by factors such as temperature, organic solvent in the reaction medium, substrate concentration, etc.

Method used

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  • A lipase mutant with improved catalytic activity and its application
  • A lipase mutant with improved catalytic activity and its application
  • A lipase mutant with improved catalytic activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of lipase mutants

[0048] like figure 1 As shown, using the site-directed mutagenesis kit, using the pPIC9K-TLL recombinant plasmid as a template, and using the following primers, site-directed mutagenesis amplification was performed. After the amplification was completed, 10 μL of PCR product was taken for agarose gel electrophoresis detection, and after verifying that the size of the band was correct, 1 μL of DMT enzyme was added to the PCR product, mixed well, and digested at 37° C. for 1 hour. Then transform: add 3 μL of digested product to 50 μL DMT competent cells, ice bath for 30 min, then heat shock in a molecular water bath at 42 °C for 45 s, ice bath for 2 min, add 500 μL of LLB medium to the product, at 37 °C, 180rpm Incubate in a shaker for 1 hour, and finally take 200 μL of bacterial solution and apply it to kan + Resistant LB dishes were incubated overnight in a 37°C incubator. On the second day, a single colony on the plate was r...

Embodiment 2

[0057] Example 2 Determination of enzyme activity and enzymatic properties of lipase mutants

[0058] 1. Determination of enzyme activity of lipase mutants

[0059] The unit of enzyme activity is defined as: the amount of enzyme required to hydrolyze the substrate p-NP per minute under certain conditions to generate 1 μmoL of p-nitrophenol is one unit of enzyme activity, expressed as U. p-Nitrophenol method: pipette 420 μL of 50 mM Tris-HCl buffer at pH 9.0 into a centrifuge tube, then add 30 μL of 10 mM substrate p-NP, mix well, warm up at 37 °C for 2 min, and then add the diluted enzyme Add 50 μL of 10% SDS to stop the reaction, and finally add 500 μL of 0.5M sodium carbonate to develop color, and measure its OD value with a microplate reader at a wavelength of 405 nm. The enzyme activity assay results of the mutants are shown in Table 1: the enzyme activities of the mutants D116K, D163F, D170A, and D259Y were increased by 35.5%, 17.0%, and 44.2%, respectively, compared wit...

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Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a lipase mutant with improved catalytic activity and its application. The amino acid sequence of the lipase mutant is as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO. 4. As shown in SEQ ID NO.5, the lipase mutant changes from aspartic acid at position 116 to lysine and aspartic acid at position 163 in the amino acid sequence SEQ ID NO.1 encoding Thermomyces lanuginosus lipase Aspartic acid is changed to phenylalanine, aspartic acid at position 170 is changed to alanine, aspartic acid at position 259 is changed to tyrosine, and its coding genes are as SEQ ID NO.6, SEQ ID NO. 7. Shown in SEQ ID NO.8 and SEQ ID NO.9. The catalytic activity of the four mutants was improved.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and relates to a lipase mutant, in particular to a lipase mutant with improved catalytic activity and application thereof. Background technique [0002] The full name of lipase (Lipase, EC 3.1.1.3) is triacylglycerol acyl hydrolase, which is widely present in organisms and belongs to carboxyl ester hydrolase. Lipase can catalyze reactions such as transesterification, esterification, alcoholysis and acidolysis, and can also catalyze the hydrolysis of glycerol ester bonds of oils and fats. Lipase is widely used in detergent, food, grease, leather, medicine and other industries. Lipases are derived from animals, plants and microorganisms. Among them, lipases derived from microorganisms have a wide variety of species, short growth cycles, and wide temperature and pH ranges, which make lipases derived from microorganisms easier to apply in industrial production. [0003] Thermomyces lanug...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19A23K20/189C12R1/84
CPCC12N9/20C12Y301/01003A23K20/189
Inventor 黄遵锡韩楠玉唐敏源姜占宝
Owner YUNNAN NORMAL UNIV
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