Construction of recombinant bacterium capable of efficiently expressing chitinase and screening of mutant with high enzyme activity

A chitinase and mutant technology, applied in the field of genetic engineering, can solve the problems of insufficient secretion and low activity of wild-type chitinase, achieve considerable industrial application value, improve flux and efficiency, and enhance hydrolysis efficiency Effect

Active Publication Date: 2021-05-25
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems mentioned above about insufficient secretion of wild-type chitinase and low activity, the present invention will further incr

Method used

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  • Construction of recombinant bacterium capable of efficiently expressing chitinase and screening of mutant with high enzyme activity
  • Construction of recombinant bacterium capable of efficiently expressing chitinase and screening of mutant with high enzyme activity
  • Construction of recombinant bacterium capable of efficiently expressing chitinase and screening of mutant with high enzyme activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of pMATE-BcchiA1 recombinant plasmid and recombinant bacteria

[0027] 1. Construction of pMATE-BcchiA1 recombinant plasmid

[0028]Chitinase BcchiA1 from Bacillus circulans WL-12 has strong affinity and catalytic activity for highly insoluble chitin. Entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to artificially synthesize the gene after optimization according to the codon preference, and add a His tag to the C-terminus for protein purification. Its nucleic acid sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO. 2, the gene was synthesized and cloned into the vector pUC57-Amp to obtain the recombinant plasmid pUC57-BcchiA1. The recombinant plasmid pUC57-BcchiA1 was used as a template, and a primer pair composed of primer FragmentF and primer FragmentR was used for PCR amplification to obtain a BcchiA1 fragment of about 1.9 kb. Using the pMATE plasmid as a vector template, the vector backbone was amplified fro...

Embodiment 2

[0034] Embodiment 2 develops the new method of directed evolution of chitinase

[0035] 1. Construction of chitinase mutation library

[0036] Using the wild-type BcchiA1 nucleic acid sequence shown in SEQ ID NO.1 as a template, through the TIANZ adjustable error-prone PCR kit, use primers Fragment-F and Fragment-R to amplify the target gene, and introduce random mutations into the BcchiA1 gene (1.9kb). The error-prone PCR reaction system is shown in Table 2. The error-prone PCR reaction program is: 94°C, 3min; 94°C for 1min, 45°C for 1min, 72°C for 2min, 50 cycles.

[0037] Table 2

[0038] Element Volume (μL) Error-prone PCRMix, 10x 3.0 dNTP for error-prone PCR, 10 3.0 MnCl for error-prone PCR 2

1.0 dGTP for error-prone PCR 0 BcchiA1 template (5ng / μL) 3.0 Fragment-F / R(10Meach) 1.0 TaqDNA polymerase for error-prone PCR 0.5 Ultra-pure water 17.5

[0039] The PCR product that has introduced random mutations...

Embodiment 3

[0042] Example 3 High enzymatic activity single mutation site further superposition-site-directed mutation

[0043] 1. Single-point mutants Obtain two-point superposition mutants through site-directed mutation

[0044] 1. The acquisition of mutant BcchiA1 (Y10A / R301A): with the single-point mutant BcchiA1 (Y10A) as a template, the primer pair consisting of primers R301A-F and primer R301A-R shown in Table 3 was used for PCR amplification to obtain BcchiA1 (Y10A / R301A) fragment of about 1.9 kb. After purification by the Gel Recovery Kit, pass the kit The completed fragment was seamlessly connected with the vector backbone pMATE in Example 1, and transformed into Escherichia coli DH5α competent cells. After sequencing, the construction of the recombinant plasmid pMATE-BcchiA1(Y10A / R301A) was completed.

[0045] 2. The acquisition of mutant BcchiA1 (Y10A / E327A): with the single-point mutant BcchiA1 (Y10A) as a template, the primer pair consisting of primers E327A-F and primers...

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Abstract

The invention relates to construction of a recombinant bacterium for efficiently expressing chitinase and screening of a mutant with high enzyme activity. According to the invention, efficient secretory expression of BcchiA1 in bacillus subtilis is realized by means of a recombinant protein technology, and the problem of insufficient chitinase secretion amount of most microorganisms is solved. Meanwhile, a novel chitinase high-throughput screening method is created, and the screening throughput and efficiency are greatly improved. In addition, the invention relates to several strains of chitinase mutants with high enzyme activity, the enzyme activities of BcchiA1 (Y10A), BcchiA1 (R301A), BcchiA1 (E327A), BcchiA1 (Y10A/R301A), BcchiA1 (Y10A/E327A), BcchiA1 (R301A/E327A) and BcchiA1 (Y10A/R301A/E327A) are respectively improved by 2.49 times, 0.67 times, 3.61 times, 2.39 times, 3.46 times, 4.75 times and 16.89 times compared with the enzyme activities of the wild type BcchiA1, and the enzyme activity of BcchiA1 (Y10A/R301A/E327A) is optimal. Finally, after the chitinase BcchiA1 and the optimal mutant BcchiA1 (Y10A/R301A/E327A) of the chitinase BcchiA1 and the monooxygenase BatLPMO10 from bacillus atrophaeus are subjected to a synergistic effect respectively, the hydrolysis efficiency of the chitinase BcchiA1 can be improved by 50.00%, and the hydrolysis efficiency of the chitinase BcchiA1 (Y10A/R301A/E327A) can be improved by 46.71%.

Description

technical field [0001] The invention relates to the construction of a highly efficient secretion and expression chitinase recombinant bacterium of Bacillus circulus and the high-throughput screening of chitinase mutants, belonging to the technical field of genetic engineering. Background technique [0002] Chitin is a linear high polymer linked by N-acetyl-D-glucosamine (NAG) through β-1,4 glycosidic bonds. It is the most widely distributed amino polysaccharide in nature, especially as arthropod exoskeleton and fungal Structural component of the cell wall. Chitin has good biocompatibility, broad-spectrum antibacterial properties, and no side effects, so it has broad application prospects in biomedicine, food, environmental protection, and cosmetics industries. However, the high crystallinity and poor solubility of chitin are undoubtedly a severe challenge to further enhance the application value. [0003] Chitooligosaccharides, with a degree of polymerization lower than 20...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/75C12N1/21G01N21/31C12R1/125
CPCC12N9/2442C12Y302/01014C12N15/75G01N21/31
Inventor 张大伟王思佳付刚
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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