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Lipase mutant with improved thermal stability and applications thereof

A technology of thermal stability and lipase, applied in the field of molecular biology, can solve the problems of lipase inactivation and denaturation, volatile inactivation, TTL instability, etc., and achieve the effect of good thermal stability and improved thermal stability.

Active Publication Date: 2018-11-30
SOUTH CHINA UNIV OF TECH
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AI Technical Summary

Problems solved by technology

[0003] Many industrial fields have certain requirements for the thermal stability of lipase. It is hoped that lipase can maintain good stability under high temperature conditions, such as feed and papermaking and other industrial fields. Many process links involve high temperature environments, and lipase with poor thermal stability In the above process, it is easy to inactivate and denature, which limits the application of lipase in these industrial fields, so it is of great significance to develop lipase with good thermal stability
In previous experiments, our research group obtained lipase TTL, which has good hydrolysis activity on medium and long-chain glyceride substrates, and acts on a wide range of substrates. However, TTL is unstable in high temperature environments and is prone to denaturation. Live (TTL enzyme activity retention rate after heat treatment at 75°C and 80°C for 30 minutes is only 25% and 16%), which is not conducive to its industrial application

Method used

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  • Lipase mutant with improved thermal stability and applications thereof
  • Lipase mutant with improved thermal stability and applications thereof
  • Lipase mutant with improved thermal stability and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, construction of lipase TTL expression vector

[0028] Analysis of the lipase TTL gene reported on the NCBI website (Genebank: JF414585.1) revealed that the full length of the TTL lipase gene was 1083 bp, including 4 exons and 3 introns. Through comparison, it was found that the full length of its open reading frame was 876bp, encoding 291 amino acids, and the first 17 amino acids at the N-terminal of the protein were signal peptides. According to the searched TTL gene sequence, the TTL gene ttl was synthesized by the whole gene synthesis technology. A pair of primers were designed according to the sequence of the synthetic gene ttl (primer sequences were fw: 5'-agtcgaattcTCTCC AGTCAGACGTGAGGTT-3' and rev: 5'-ttctctagaTTACAAACAAGTACCAATCAA-3') to amplify the mature peptide gene tll- 1. Cloning the amplified tll-1 into the vector pPICZαA to obtain the recombinant vector pPICZαA-ttl-1.

Embodiment 2

[0029] Embodiment 2, construction disulfide bond mutant

[0030] First, the three-dimensional protein structure of TTL was obtained through the homology modeling software Modeller, and then the molecular dynamics simulation of TTL was performed through the molecular dynamics software Gromacs, and the flexible region of the TTL protein was found according to the results of the molecular dynamics simulation. Add disulfide bonds in flexible regions of the protein. Five pairs of disulfide bonds were designed through the online disulfide bond design website (http: / / cptweb.cpt.wayne.edu / DbD2 / index.php), namely E61C / N31C, E61C / T69C, I108C / A162C, G166C / G195C and G230C / V233C.

[0031] The construction of the disulfide bond mutant is roughly as follows (taking the mutant E61C / N31C as an example, and so on): using the constructed pPICZαA-ttl-1 as a template, first use the upstream and downstream primers E61C-fw and E61C-rev to carry out PCR amplification, agarose electrophoresis to de...

Embodiment 3

[0034] Example 3, Screening of Disulfide Bond Mutant Recombinant Yeast Engineering Strains

[0035] Pick the yeast recombinant transformants in Example 2 one by one with a toothpick to a 24-well plate containing 2 mL of BMGY medium in each well, culture at 30°C and 220 rpm for about 36 hours, and then add 0.75% (v / v) methanol to induce nourish. After culturing at 30° C. and 220 rpm for 24 hours, the supernatant was collected by centrifugation for enzyme activity determination. The lipase activity was determined by acid-base titration, and the instrument used was a pH-stat acid-base titrator (Swiss Metrohm China Co., Ltd.). The specific steps of enzyme activity determination are as follows: first, 5% (v / v) olive oil is added to 100mL water, and 2% (w / v) gum arabic is added as a stabilizer at the same time, and the olive oil substrate is obtained after high-speed homogenization for 5 minutes ; Take 15mL of olive oil substrate and add it to the reaction cup. After the reaction ...

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Abstract

The invention discloses a lipase mutant with improved thermal stability and applications thereof, wherein the lipase mutant is obtained by adding a disulfide bond to a lipase TTL, the nucleotide sequence of the lipase TTL is represented by SEQ ID NO.1, and the amino acid sequence is represented by SEQ ID NO.2. According to the present invention, by constructing the disulfide bond, the lipase mutant TTL-108 / 162 with improved thermal stability is obtained; and the enzyme activity retention rates of the mutant TTL-108 / 162 after the heat treatments for 30 min at temperatures of 75 DEG C and 80 DEGC respectively are 65% and 48%, such that the obtained lipase mutant TTL-108 / 162 of the present invention has good thermal stability and can be widely used in the industrial field.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a lipase mutant with improved thermostability and application thereof. Background technique [0002] Lipase is a general term for a class of enzymes that catalyze the decomposition or synthesis of glycerides at the oil-water interface, which can catalyze the hydrolysis of natural substrate oils and release glycerides or glycerol and fatty acids with fewer ester bonds. At the same time, it can catalyze reactions such as acidolysis, transesterification, ester synthesis and transesterification. Due to the unique enzymatic characteristics of lipase, it has wide application potential in the fields of feed and paper making. [0003] Many industrial fields have certain requirements for the thermal stability of lipase. It is hoped that lipase can maintain good stability under high temperature conditions, such as feed and papermaking and other industrial fields. Many process links involv...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12R1/84
CPCC12N9/20C12N15/815C12Y301/01003
Inventor 杨博王建荣王永华张天宇吴宗泽蓝东明赵格
Owner SOUTH CHINA UNIV OF TECH
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