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Lipase calb mutant, its preparation method and application

A mutant and lipase technology, applied in the field of genetic engineering, can solve the problems of enzyme instability, insufficient enzyme source, substrate water insolubility, etc., and achieve the effect of mass production and high expression activity

Active Publication Date: 2018-09-21
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although several decades have been studied in the breeding of lipase-producing strains, culture conditions, properties of enzymes, and industrial applications, due to the diversity of structures and properties of lipases, the instability of enzymes, the water insolubility of substrates, Insufficient sources of enzymes, difficult purification, and limited application range, the research progress and industrial application of lipase are much inferior to those of protease and amylase

Method used

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  • Lipase calb mutant, its preparation method and application
  • Lipase calb mutant, its preparation method and application
  • Lipase calb mutant, its preparation method and application

Examples

Experimental program
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Embodiment 1

[0026] Embodiment 1 constructs lipase CALB mutant library

[0027] 1. The coding gene calb of CALB is amplified by lipase-producing Klebsiella sp.;

[0028] 2. The calb gene was connected to the expression plasmid pPICZαA, and the restriction sites were EcoRI and XbaI to obtain the recombinant plasmid pPICZαA-calb;

[0029] 3. Using error-prone PCR technology, using the recombinant plasmid pPICZαA-calb as a template, design primers to amplify and obtain the calb mutant gene;

[0030] 4. The error-prone PCR product obtained in step 3 and the expression plasmid pPICZαA were digested with EcoRI and XbaI and ligated, and the ligated product was transferred into Pichia pastoris X33 competent cells, and coated on bleomycin-resistant (Zec + ) on the YPD plate to obtain the constructed recombinant calb gene mutation library;

[0031] 5. The colonies grown on the YPD plate were transferred to methanol-containing tributyrin (Zec + ) plate, after transferring the plate, place the plat...

Embodiment 2

[0032] The acquisition of embodiment 2 lipase CALB mutant gene

[0033] Using 1 μg of the high lipase-producing strain genomic DNA of Example 1 as a PCR reaction template, the forward primer calb-F: 5′-AAAAAGAATTCAACAAACACGTCGCTGCTATGCTGACGATGCTTATTA-3′, the reverse primer calb-R: 5′-AAAAATCTAGAGTGGTGGTGGTGGTGGTGTCTTTGAGATTTTGGTCTAAAAAA-3′ were designed, wherein The parts in italics are the enzyme cutting sites EcoRI and XbaI, respectively. In order to ensure the correct reading frame, an additional five bases were added to the 5' ends of the forward and reverse primers. The PCR reaction was carried out in a total volume of 50 μL, and the reaction conditions were as follows: denaturation at 94°C for 5 minutes followed by cycling at 94°C for 50 s, annealing at 58°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles, and extension at 72°C for 10 minutes. Take 3 μL of PCR amplification products for agarose gel electrophoresis verification, the results are as foll...

Embodiment 3

[0034] The construction of embodiment 3 expression vector pET-28b (+)-calb

[0035] Gel recovery The PCR product of Example 2 and the pPICZαA vector were double-digested with EcoRI and XbaI respectively, and recovered by a gel recovery kit, then ligated (16°C, 16h), transformed into DH5α competent cells, and positive clones were selected. After extracting the plasmid, it was verified by enzyme digestion analysis, and the results were as follows: figure 2shown, and carried out DNA sequencing identification, the nucleotide sequence encoding the lipase CALB mutant is shown in SEQ ID No.2, and the amino acid sequence of the CALB mutant is shown in SEQ ID No.1. The constructed expression plasmid was called pPICZαA-calb.

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Abstract

The invention provides lipase CALB mutant and a preparation method and application thereof. An error-prone PCR technology in a gene in-vitro directed evolution strategy is utilized, mutagenesis in-vitro is conducted on a wild type lipase gene from Klebsiella sp., screening is conducted by distinguishing a culture medium, and a strain for efficiently producing lipase is obtained. The obtained lipase CALB mutant is high in expression activity, the expression activity is 8.7 times catalytic activity of the wild type lipase, and the lipase CALB mutant can be used for industries related to lipid hydrolysis. Batched production of the lipase CALB mutant can be achieved through large-scale fermentation of engineering bacteria containing the gene for encoding the CALB mutant.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a lipase CALB mutant, its preparation method and application. Background technique [0002] Lipase (Lipase, glyceride hydrolase) is an enzyme that breaks down fat. Ubiquitous in animals, plants and microorganisms, it is a special type of ester bond hydrolase that catalyzes the following reaction: triglyceride + water → glycerol + free fatty acid. Another important feature of lipase is that it only acts on a heterogeneous system, that is, it acts on an oil (or fat)-water interface, and has no effect on a uniformly dispersed or water-soluble substrate, even if it acts very slowly, so it is also It can be said that lipase is an enzyme that specifically hydrolyzes esters at the oil (fat)-water interface of heterogeneous systems or water-insoluble systems. Lipase is one of the earliest studied enzymes. From the report of rabbit pancreatic lipase activity in 1834 to now, the resear...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19C11D3/386C12R1/84
CPCC11D3/38627C12N9/20
Inventor 汪本助杨为华穆晓玲徐斌张雪锋
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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