Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Lipase mutant of marine streptomyces and application of lipase mutant

A technology of marine streptomyces and lipase, applied in the fields of genetic engineering and enzyme engineering, can solve problems such as difficulty in screening ideal strains, poor enzymatic properties, and small workload

Active Publication Date: 2019-12-06
SOUTH CHINA UNIV OF TECH
View PDF8 Cites 9 Cited by
  • 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

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Lipase mutant of marine streptomyces and application of lipase mutant

Examples

Experimental program
Comparison scheme
Effect test

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 measured. 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 single ...

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 ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a lipase mutant of marine streptomyces and application of the lipase mutant, and belongs to the technical field of genetic engineering and enzyme engineering. According to thepresent invention, an enzyme mutation library is designed and constructed based on the structure analysis of an enzyme protein, the lipase mutant preferring to a substrate of partial glycerides is screened and obtained, and a Pichia engineering strain that efficiently expressed the mutant is constructed and obtained, so that a foundation for the wide application of the mutant is laid. Compared with the wild-type lipase, the lipase mutant of marine streptomyces provided by the invention is more efficient in catalyzing the synthesis of partial glycerides rich in n-3 fatty acids. Under the optimal reaction conditions, the content of partial glycerides in the catalytic product is 86.48% (49.01% of PUFA-DAG, and 37.47% of PUFA-MAG), and is increased to about 23 times compared with the wild-typelipase (3.8% of PUFA-DAG).

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com