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Novel lipase as well as gene, engineering bacteria and preparation method thereof

A lipase and gene technology, applied in the fields of novel lipase mutants and their preparation and application, can solve the problems of short half-life, increased industrial application cost, poor thermal stability, etc., and achieve the effect of high-efficiency expression

Inactive Publication Date: 2017-11-21
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the lipases used in industry are mesophilic enzymes with poor thermal stability and a very short half-life at high temperatures
This not only limits its application range, but also increases the cost of industrial applications, which brings difficulties to practical applications.

Method used

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  • Novel lipase as well as gene, engineering bacteria and preparation method thereof
  • Novel lipase as well as gene, engineering bacteria and preparation method thereof
  • Novel lipase as well as gene, engineering bacteria and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Obtaining wild-type lipase gene

[0064] 1. The wild-type lipase gene was derived from Penicillium cyclopium CICC 41049 strain, and its total RNA was extracted.

[0065] (1) Strain activation: draw 100 μL of the preserved Penicillium arcuate spore liquid from the glycerol tube, spread it evenly in the PDA eggplant bottle, and culture at 28°C for 5 days;

[0066] (2) Transfer: Wash the spores in the eggplant bottle with sterile water, centrifuge at 12000r / min for 1min, repeatedly wash, and finally transfer to 50mL PDA liquid medium, place in a shaker, 28℃, 200r / min, culture for 2d;

[0067] (3) Collect the bacteria: use double-layer sterilized gauze, filter the bacteria, rinse with sterile water, wring dry, place the bacteria in a mortar, add liquid nitrogen and grind to powder;

[0068] (4) Adding Trizol: Dispense a small amount of the ground powder into EP tubes, add 1 mL of LTrizol reagent to each tube, shake at room temperature for 10 minutes, and place on ice for...

Embodiment 2

[0094] Example 2: Construction of recombinant heat-resistant lipase of E. coli

[0095] 1. The wild-type lipase gene is ligated with pET-22b(+) vector.

[0096] The purified pcl was ligated with the pET-22b(+) vector, and then the recombinant plasmid was transformed into E. coli DH 5α, and the wild-type lipase gene was successfully cloned into pET- by double digestion with BamH I and Hind III. The recombinant plasmid pET-pcl was constructed on the 22b(+) vector.

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

[0098] ddH 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

dTTP(10mmol / L)

5μL

dCTP(10mmol / L)

5μL

MgCl 2 (25mmol / L)

10μL

MnCl 2 (10mmol / L)

1.25μL

[0099] After the system is completed, carry out the erro...

Embodiment 3

[0111] Example 3: Construction of Bacillus subtilis heat-resistant lipase recombinant bacteria

[0112] 1. Construction of expression vector pBSA43

[0113] Using Escherichia coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloned into a strong Bacillus constitutive promoter P43 (SEQ ID No. 7) and levansucrase that can secrete the recombinant protein directly into the medium The signal sequence sacB (SEQ ID No. 8) obtained the expression vector pBSA43. It comes with Amp r And Kana r Genes can use ampicillin resistance as a selection marker in Escherichia coli, while kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0114] 2. Construction of the heat-resistant lipase expression vector pBSA43-mpcl 3

[0115] The heat-resistant lipase gene constructed by error-prone PCR and the Bacillus subtilis expression vector pBSA43 were both digested with BamHI and HindIII, and then ligated to construct a recombinant plasmid...

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Abstract

The invention belongs to the technical field of gene engineering of enzyme, and particularly relates to novel lipase as well as a gene, engineering bacteria and a preparation method thereof. A wild-type lipase gene is obtained by virtue of a molecular biology technical means, after a recombinant vector is established by virtue of digestion, connection and the like, an error-prone PCR technique is used for randomly mutating the wild-type lipase gene to obtain a lipase mutant G47I and a coded gene mpcl3 thereof, the recombinant vector is re-established, so that the high-efficient expression in bacillus subtilis WB600 and pichia pastoris GS115 can be realized, and heat-resistant lipase can be obtained by virtue of techniques such as fermentation and extraction.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a novel lipase mutant and its preparation and application. Background technique: [0002] Lipase (Lipase, EC3.1.1.3), the full name is triacylglyce-rolacylhydrolase (triacylglyce-rolacylhydrolase), can hydrolyze triacylglycerides between water-insoluble substrate and water interface to generate diglycerides and monoglycerides, It also includes a series of biological reactions such as esterification, transesterification, alcoholysis, acidolysis, and aminolysis. Lipase is widely distributed in nature, and lipase is found in animals, plants and microorganisms. Lipase derived from microorganisms has more advantages over animals and plants: easy to cultivate, short production cycle and high yield, which is convenient for industrialized production; it has a wide range of sources. Due to the large number and different types of microbial lipase, it has a broade...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/75C12N15/81C12N1/21C12N1/19C12R1/125C12R1/84
CPCC12N9/20C12N15/75C12N15/815C12Y301/01003
Inventor 刘逸寒路福平刘浩郑东
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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