Novel lipase as well as preparation and application thereof

A lipase and mutant technology, applied in the direction of application, enzyme, hydrolase, etc., to achieve high-efficiency expression

Active Publication Date: 2019-04-05
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some of the lipases that have been discovered have shown a certain degree of alkali toler...

Method used

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  • Novel lipase as well as preparation and application thereof
  • Novel lipase as well as preparation and application thereof
  • Novel lipase as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: the acquisition of wild-type lipase gene

[0062] 1. The wild-type lipase gene is from Penicillium cyclopium CICC 41049 strain, and its total RNA is extracted.

[0063] (1) Strain activation: draw 100 μL of preserved Penicillium arcuci spore liquid from the glycerol tube, evenly spread it in the PDA eggplant bottle, and incubate at a constant temperature of 28°C for 5 days;

[0064] (2) Transfer: Wash the spores in the eggplant bottle with sterile water, centrifuge at 12000r / min for 1min, wash repeatedly, and finally transfer to 50mL PDA liquid medium, place in a shaker, 28°C, 200r / min min, cultivated for 2 days;

[0065] (3) Collect the bacteria: filter the bacteria with double-layer sterilized gauze, rinse with sterile water, wring out, place the bacteria in a mortar, add liquid nitrogen and grind to powder;

[0066] (4) Add Trizol: Take a small amount of the ground powder and put them into EP tubes, add 1mL Trizol reagent into each tube, shake the nes...

Embodiment 2

[0094] Embodiment 2: Obtaining of lipase mutant N157F

[0095] 1. Ligate the codon-optimized wild-type lipase gene of Escherichia coli to the pET-22b(+) vector.

[0096] The purified pcl was ligated with the pET-22b(+) vector, and then the recombinant plasmid was transformed into Escherichia coli DH 5α, and the codon-optimized wild-type fat of Escherichia coli was successfully verified by double digestion with BamH I and Hind III The enzyme gene has been cloned into the pET-22b(+) vector to construct the recombinant plasmid pET-pcl.

[0097] 2. Error-prone PCR: Using the recombinant plasmid pET-pcl constructed above as a template, the reaction system is as follows:

[0098] wxya 2 o

21μL

Recombinant plasmid pET-pcl (5ng / μL)

1μL

Upstream primer P1 (10μmol / L)

2μL

Downstream primer P2 (10μmol / L)

2μL

Taq DNA polymerase

0.5μL

10×Taq buffer

5μL

dATP (10mmol / L)

1μL

dGTP (10mmol / L)

1μL

...

Embodiment 3

[0112] Embodiment 3: the construction of the lipase recombinant bacterium that Bacillus subtilis alkali resistance improves

[0113] 1. Construction of expression vector pBSA43

[0114] Using the Escherichia coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, clone into a strong Bacillus constitutive promoter P43 (SEQ ID No.10) and fructan sucrase that can directly secrete the recombinant protein into the medium The signal sequence sacB (SEQ ID No.11) was obtained from the expression vector pBSA43. it comes with amp r and Kana r Gene, ampicillin resistance can be used as a selection marker in Escherichia coli, and kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0115] 2. Construction of lipase expression plasmid pBSA43-Bsmpcl with improved alkali resistance

[0116] The codon-optimized lipase mutant gene Bsmpcl (SEQ ID No.6) of Bacillus subtilis and the expression vector pBSA43 of Bacillus subtilis w...

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Abstract

The invention belongs to the technical field of gene engineering of enzyme, and specifically relates to a lipase mutant of which the alkaline resistance is increased, as well as preparation and application thereof. A wild type lipase gene is obtained through a molecular biology technological means, and random mutation is carried out on the wild type lipase gene subjected to escherichia coli codonoptimization by utilizing an error-prone PCR (Polymerase Chain Reaction) technology, so that a lipase mutant N157F and an encoding gene mpc1 thereof can be obtained; a recombinant vector can be reconstructed, efficient expression of the recombinant vector in bacillus subtilis WB600 and pichia pastoris GS115 is realized, and the lipase mutant of which the alkaline resistance is further increased isobtained through technologies of fermentation, extraction and the like.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme genetic engineering, and in particular relates to a lipase mutant with improved alkali resistance and its preparation and application. Background technique: [0002] Lipase (Lipase, E.C.3.1.1.3), the full name of triacylglycerol hydrolase (triacylglyce-rolacylhydrolase), belongs to carboxyl ester hydrolase, it can catalyze the hydrolysis of natural substrate oil to generate fatty acid, glycerol and monoglycerol Lipids are mainly distributed in animal, plant and microbial tissues. The low content and activity of lipase in animals will result in a low level of industrial production. Lipase in plants mainly exists in oil-rich oilseeds. Because of the large difference in enzyme activity, there are relatively few studies on plant lipase. Due to the variety of microorganisms and rapid reproduction, they have a wider range of reaction pH, reaction temperature and substrate selectivity, and microbial li...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/75C12N15/81
CPCC12N9/20C12N15/75C12N15/815C12Y301/01003
Inventor 刘逸寒路福平马杰莹邵舒琳
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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