Glyceride lipase mutant G28C-P206C as well as coding gene and application thereof

A G28C-P206C, lipase technology, applied in the field of enzyme engineering, can solve problems such as unfavorable industrialization, limited application scope, easy inactivation, etc.

Active Publication Date: 2021-03-30
SOUTH CHINA UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A glyceride lipase SMG1 derived from Malassezia globosa was screened in the laboratory in the early stage, which can be used for the synthesis of high-purity diglycerides, oil deacidification and epoxidation reactions, etc., but found in use that its thermal The stability is poor, and it is easy to be inactivated during the application process, resulting in an increase in production costs, limiting the scope of application, and is not conducive to industrialization

Method used

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  • Glyceride lipase mutant G28C-P206C as well as coding gene and application thereof
  • Glyceride lipase mutant G28C-P206C as well as coding gene and application thereof
  • Glyceride lipase mutant G28C-P206C as well as coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the construction of glyceride mutant lipase expression vector

[0041] The expression vector pPICZaA-SMG1 of glyceride lipase SMG1 was constructed in the early stage of this experiment, that is, the gene of SMG1 (Genbank ID: XM_001732152.1) was inserted into the vector pPICZaA, and the restriction sites were EcoRI and SalI. Analyzing the crystal structure of lipase SMG1 (PDB ID: 3UUE), based on the experience accumulated by the inventors, and using Disulfide byDesign software to predict potential disulfide bond mutation sites, after repeated comparisons by the inventors, 5 disulfide bonds were finally left Key mutants, i.e. glyceride lipase mutant T74C-N85C, glyceride lipase mutant Y127C-L186C, glyceride lipase mutant N38C-A257C, glyceride lipase mutant G28C-P206C or glyceride lipase mutant Body T42C-F286C. Each disulfide bond mutant involves a combination of two mutation sites, that is, two amino acid residues are mutated into Cys, and the two Cys will l...

Embodiment 2

[0052] Embodiment 2: Construction, expression and purification of glyceride lipase mutant expression strain

[0053] After the positive transformants with correct sequencing were amplified overnight in LLB liquid medium, the plasmids were extracted, linearized with PmeI, purified and recovered, and transformed by electroporation with a total of 5 μg of plasmid linearized products mixed with X33 Pichia pastoris competent . Competent preparation of Pichia pastoris refers to the operation manual of Invitrogen Company. The electroporation program was set according to the parameters recommended by Bio-Rad.

[0054] Add 1 mL of 1mol / L sorbitol solution immediately after electroporation, incubate and recover the bacterial solution at 30°C for 1 hour, and spread it evenly on the YPDS+Zeocin (Zeocin concentration is 100 μg / ml) resistance plate for screening; after culturing for 72 hours, select positive Turn.

[0055] A single colony of the engineering strain was inoculated into 50 ...

Embodiment 3

[0057] Example 3: DSF Screening of Glyceride Lipase Mutants

[0058] Dilute the target protein to the same concentration of 0.3mg / ml, and dilute the dye Sypro Orange dye 100 times. Mix 20 μL of protein with 5 μL of dye, and perform protein thermal inactivation curve measurement in Bio-Rad’s real-time fluorescent quantitative PCR instrument to obtain the T of the protein. m value. The results are shown in Table 2, the T of glyceride lipase mutant G28C-P206C m It is 9.0°C higher than the wild type and 3.0°C higher than the mutant S5.

[0059] Table 2 DSF measurement results

[0060]

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Abstract

The invention discloses a glyceride lipase mutant G28C-P206C as well as a coding gene and application thereof. According to the glyceride lipase mutant G28C-P206C, a disulfide bond is introduced intoglyceride lipase SMG1, the amino acid sequence of the mutant is shown as SEQ ID NO.3, and the nucleotide sequence of the mutant is shown as SEQ ID NO.4. Compared with a wild type, the Tm of the glyceride lipase mutant G28C-P206C obtained by the invention is increased by 9.0 DEG C, the half-life period at 50 DEG C is increased by 64.8 times, the thermal stability is remarkably improved, and compared with the wild type, the glyceride lipase mutant G28C-P206C has better enzymatic activity and is more suitable for application in the industrial field.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a glyceride lipase mutant G28C-P206C and its coding gene and application. Background technique [0002] Lipase (EC 3.1.1.3) is a serine hydrolase that not only hydrolyzes oil at the oil-water interface to produce products such as fatty acids, diglycerides, monoglycerides, and glycerol, but also catalyzes esterification, transesterification, alcoholysis, and acid Solution and other reactions. Triglyceride lipase can hydrolyze or esterify to form triglyceride, diglyceride or monoglyceride, while glyceride lipase can only decompose diglyceride or monoglyceride, but not triglyceride. The esterification involved The reaction also synthesizes only diglycerides or monoglycerides. Glyceride lipase plays an important role in the preparation of new structural lipids and the removal of glycerides in oils, so it has great market prospects in the fields of oil processing, medic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12P7/64C12R1/84
CPCC12N9/20C12Y301/01003C12P7/6463C12N15/815
Inventor 王永华李力浪蓝东明杨博
Owner SOUTH CHINA UNIV OF TECH
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