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Lipase mutant D163F with improved catalytic activity and application thereof

一种催化活性提高、D163F的技术，应用在酶工程领域，能够解决不能满足工业需求等问题

Active Publication Date: 2021-03-30
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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  • Lipase mutant D163F with improved catalytic activity and application thereof
  • Lipase mutant D163F with improved catalytic activity and application thereof
  • Lipase mutant D163F with improved catalytic activity and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Preparation of embodiment 1 lipase mutant

[0049] Such as figure 1 As shown, using the site-directed mutagenesis kit, the pPIC9K-TLL recombinant plasmid was used as a template, and the following primers were used for site-directed mutagenesis amplification. After the amplification is completed, take 10 μL of the PCR product for agarose gel electrophoresis detection. After verifying that the band size is correct, add 1 μL of DMT enzyme to the PCR product, mix well, and digest at 37°C for 1 hour. Then transform: add 3 μL of digested product to 50 μL of DMT competent cells, ice bath for 30 minutes, then heat shock in 42 °C molecular water bath for 45 seconds, ice bath for 2 min, add 500 μL LB medium to the product, at 37 °C, 180 rpm Incubate in a shaker for 1 hour, and finally take 200 μL of bacterial liquid and apply it to kan + Resistant LB dishes were cultured overnight in a 37°C incubator. On the second day, a single colony on the plate was randomly selected for po...

Embodiment 2

[0058] Embodiment 2 lipase mutant enzyme activity and the mensuration of enzymatic properties

[0059] 1. Determination of lipase mutant enzyme activity

[0060] The enzyme activity unit is defined as: under certain conditions, the amount of enzyme required to hydrolyze the substrate p-NP per minute to generate 1 μmoL of p-nitrophenol is expressed as an enzyme activity unit expressed in U. p-Nitrophenol method: Pipette 420 μL of 50 mM Tris-HCl buffer solution with a pH of 9.0 into a centrifuge tube, then add 30 μL of 10 mM substrate p-NP, mix well, preheat at 37°C for 2 minutes, and then add the diluted 50 μL of enzyme solution was reacted for 5 minutes, 50 μL of 10% SDS was added to terminate the reaction, and finally 500 μL of 0.5 M sodium carbonate was added for color development, and the OD value was measured with a microplate reader at a wavelength of 405 nm. The results of the enzyme activity determination of the mutants are shown in Table 1: the enzyme activities of th...

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Abstract

The divisional application discloses a lipase mutant D163F with improved catalytic activity and application thereof, the amino acid sequence of the lipase mutant D163F is shown as SEQ ID NO.3, the lipase mutant D163F is obtained by converting 163-site aspartic acid in the amino acid sequence SEQ ID NO.1 of encoded thermomyces lanuginosus lipase into phenylalanine, and the encoding gene of the lipase mutant D163F is shown as SEQ ID NO.7. The catalytic activity of the mutant is improved.

Description

[0001] This application is a divisional application with the application number 201910523658.3, the application date of June 17, 2019, and the title of the invention "a lipase mutant with improved catalytic activity and its application". technical field [0002] The invention belongs to the technical field of enzyme engineering and relates to a lipase mutant, in particular to a lipase mutant D163F with improved catalytic activity and application thereof. Background technique [0003] The full name of lipase (Lipase, EC3.1.1.3) is triacylglycerol acyl hydrolase, which widely exists 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 glyceride bonds in oils and fats. Lipase is widely used in detergent, food, oil, leather, medicine and other industries. Lipases are derived from animals, plants and microorganisms, among which lipases d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N1/19A23K20/189C12R1/84
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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