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A kind of marine streptomyces lipase mutant and its application

A marine Streptomyces and lipase technology, applied in the field of genetic engineering and enzyme engineering, can solve the problems of small workload, large workload, strong randomness, etc.

Active Publication Date: 2021-06-11
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The breeding of microbial strains is one of the most commonly used and simplest methods for industrial production and academic research; however, its disadvantages are heavy workload and strong randomness; therefore, it is often difficult to screen for ideal strains
Protein engineering is a method that has emerged in recent years, using methods of molecular biology and bioinformatics to carry out directed mutation and rational transformation of proteins to obtain ideal proteins or enzymes. Its advantages are relatively small workload and high probability; disadvantages The protease properties of most mutants have not changed or become worse

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  • A kind of marine streptomyces lipase mutant and its application

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Embodiment 1

[0024] Construction and expression of embodiment 1 marine Streptomyces lipase mutant

[0025] Structural analysis of MAS1 lipase (the amino acid sequence is SEQ ID NO: 1) found that, in addition to the catalytic triplet (S109-H232-D200), its catalytic pocket consists of T38, F39, G49, H108, T141, V202, V203, T237 Amino acid composition. In order to enhance the steric effect of the pocket on the TAG substrate and change the substrate selectivity of lipase, single mutants T38R / F, G40E / F, H108W, T141F / R, V202F, V203L / F, V233F / R, T237Y and double mutations H108W-T38R / F, H108W-G40F, H108W-T141F / R, H108W-V202L / F, H108WV233R / F, H108W-T237Y and other series of enzyme mutants. The genes of the above mutants were cloned into pET22b vector and transformed into BL21(DE3) for expression.

Embodiment 2

[0026] The screening of embodiment 2 marine Streptomyces lipase mutants

[0027] MAS1 lipase mutants were prepared using E. coli expression system, and emulsified olive oil (TAG: 98%, DAG: 2%) and Kao diglyceride (TAG: 65%, DAG: 32%, MAG: 2%) As a substrate, the difference in enzyme activity of the lysate was determined. The result is as figure 1 shown, from figure 1 It can be seen that the mutations at T38 and T141 lead to the loss of MAS1 lipase activity, and the mutations at V202 and V203 also greatly reduce the enzymatic hydrolysis activity. Only single mutations G40E / F, H108W, V233F, T237Y and double mutations H108W-G40E / F, H108W-V233F, H108W-T237Y had no obvious effect on the activity of the enzyme. Among them, the hydrolysis activity of G40F mutant to DAG substrate was higher than that of triglyceride substrate, suggesting that the mutation of G40F site may lead to the change of substrate selectivity of MAS1 lipase.

[0028] The amino acid sequence of the G40F singl...

Embodiment 3

[0030] Embodiment 3 Expression preparation of marine Streptomyces lipase mutant

[0031] (1) Construction of Pichia pastoris expression engineering bacteria: the MAS1-G40F gene was cloned into the Pichia pastoris expression vector pPICZαA vector, screened and sequenced to verify that the gene was correct. The pPICZαA-MAS1-G40F vector was linearized and electrotransformed into Pichia pastoris X-33 strain, and screened with bleomycin to obtain positive recombinant expression strains.

[0032] (2) Preparation of primary seed liquid: Inoculate the single colony obtained above into a sterilized Erlenmeyer flask containing 50mL of YPD liquid medium, and then place the Erlenmeyer flask at a temperature of 30°C and a rotating speed of 200rpm Vibrate in a shaking table for 18-24 hours to obtain the first-grade seed liquid;

[0033] (4) Preparation of the secondary seed liquid: Inject 5 mL of the primary seed liquid into 100 mL of sterilized fresh YPD liquid medium, and vibrate for 24 ...

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Abstract

The invention discloses a marine Streptomyces lipase mutant and its application, and belongs to the technical field of genetic engineering and enzyme engineering. The invention designs and constructs an enzyme mutation library based on enzyme protein structure analysis, and screens to obtain partial glyceride substrate preference lipase mutants, and construct Pichia pastoris engineering strains that highly express the mutants, thus laying the foundation for the wide application of the mutants. Compared with the wild-type lipase, the Streptomyces marine lipase mutant provided by the present invention catalyzes and synthesizes partial glycerides rich in n-3 fatty acids more efficiently, and under optimal reaction conditions, the partial glycerides in the catalyzed product The content of PUFA-DAG was 86.48% (including 49.01% PUFA-DAG, 37.47% PUFA-MAG), about 23 times higher than that of the wild type (PUFA-DAG 3.8%).

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and enzyme engineering, in particular to a marine Streptomyces lipase mutant and application thereof. Background technique [0002] Lipase, triacylglycerol acyl hydrolase, catalyzes the hydrolysis of natural substrate oils to produce fatty acids, glycerol and mono- or di-glycerides. The basic unit of lipase is only amino acids, usually only one polypeptide chain. Its catalytic activity depends solely on its protein structure (Schmid et al., 1998). Lipase has a wide range of applications and has become the third largest industrial enzyme on the market. Lipase can catalyze reactions such as lipolysis, transesterification, and ester synthesis, and is widely used in industries such as feed additives, oil processing, food, medicine, and daily chemicals. [0003] Enzymatic properties and enzyme stability are one of the key factors affecting its application; at the same time, many protein...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C12P7/64C12R1/84
CPCC12N9/20C12N15/815C12P7/6454C12Y301/01003
Inventor 王永华蓝东明赵泽鑫杨博
Owner SOUTH CHINA UNIV OF TECH
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