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Chitin enzyme mutant capable of improving enzyme activity

A technology of chitinase and mutant, applied in the field of enzyme engineering and genetic engineering, can solve the problems of difficult screening, long culture period, loss of enzyme activity, etc.

Active Publication Date: 2019-03-19
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there have been studies trying to increase the expression of chitinase through heterologous expression, enzyme molecular modification and other technical means, for example, heterologous expression of chitinase gene in E. However, heterologous expression of chitinase gene in Escherichia coli is easy to form inclusion bodies; heterologous expression of chitinase gene in Escherichia coli and Pichia pastoris When extracting the chitinase gene, the chitinase is secreted intracellularly. Therefore, the extraction of the chitinase requires breaking the wall to cause loss of enzyme activity; the operation of heterologously expressing the chitinase gene in Pichia pastoris is complicated and the culture period is long; the method of random mutation Modification of the substrate-binding domain and catalytic domain of chitinase to improve enzyme activity has the uncertainty of random mutation, therefore, it will lead to difficulties in screening, and the above-mentioned technologies cannot be really applied to industrial production
[0006] Therefore, it is urgent to find a new method that can overcome the defects of low expression, easy formation of inclusion bodies, and loss of enzyme activity caused by wall breaking, and can greatly increase the expression of chitinase.

Method used

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  • Chitin enzyme mutant capable of improving enzyme activity
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  • Chitin enzyme mutant capable of improving enzyme activity

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1 produces chitinase bacterial strain construction

[0030] The chitinase gene fragment chisb (nucleotide sequence shown in SEQ ID NO.9) was chemically synthesized. The PCR conditions were: 98° C. for 3 min, 30 cycles (98° C. for 10 s, 55° C. for 15 s, 72° C. for 1 min), and 72° C. for 5 min. Using pP43NMK as a template and p43-F and p43-R as primers (see Table 1) for PCR amplification of the whole plasmid, a linearized pP43NMK vector was obtained. The PCR conditions were: 98°C for 5min, 25 cycles (98°C for 10s, 55°C for 15s, 72°C for 4min and 30s), 72°C for 5min. After the above amplified products were checked by electrophoresis, the PCR products were purified and recovered using a gel recovery kit. One-step cloning kit clonExpress TM One Step Cloning Kit (Vazyme Biotech Co., Ltd. Nanjing, China), fused the chitinase gene fragment with the linearized vector pP43NMK to obtain the recombinant plasmid pP43NMK-chisb.

[0031] Transform the fused recombinant...

Embodiment 2

[0035] The verification of embodiment 2 chitinase production strains

[0036] The plasmid pP43NMK-chisb sequenced correctly in Example 1 was transformed into Bacillus.subtilis WB600. Selected transformants were inoculated into LB medium, cultured at 37°C for 8 hours; transferred to TB medium with an inoculum size of 2%, cultured at 37°C for 12 hours, collected the fermentation supernatant, and detected the enzyme activity of the fermentation supernatant, the results showed , chitinase was secreted to the outside of the cell, and the enzyme activity was 28.98U / mL (results such as figure 1 shown).

Embodiment 3

[0037] The acquisition of embodiment 3 mutant strains

[0038]Use the site-directed mutagenesis kit (purchased from TaKaRa, product number: KM101), design primers (see Table 2), and use the constructed pP43NMK-chisb as a template to carry out PCR, and the 43rd position near the catalytic domain of the chitinase molecule Cysteine ​​is mutated to aspartic acid; 273rd phenylalanine is mutated to tryptophan; or 336th glutamic acid is mutated to arginine, respectively named C43D, F273W, E336R. The PCR reaction conditions were 98°C for 5min, 30 cycles (98°C for 10s, 55°C for 15s, 72°C for 4min30s), and 72°C for 5min. The gel recovery kit was used to purify and recover the PCR products, and the recovered products were checked by electrophoresis. The product was transformed into E.coli JM109 and sequenced by Tianlin (Wuxi) Co., Ltd., and the transformants were named pP43NMK-chisb-C43D, pP43NMK-chisb-F273W, and pP43NMK-chisb-E336R.

[0039] Using the plasmid of the correctly sequence...

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Abstract

The invention discloses a chitin enzyme mutant capable of improving enzyme activity and belongs to the technical field of genetic engineering and enzyme engineering. By virtue of a site-specific mutagenesis manner, the cysteine at the site 43 nearby a chitin enzyme molecular catalysis structural domain is mutated into aspartic acid, the phenylalanine at the site 273 is mutated into tryptophan, andthe glutamic acid at the site 336 is mutated into arginine, so that the enzyme activity of the chitin enzyme expressed by the strain is obviously improved. Compared with an original strain, single mutant strains F274W and E336R have the advantages that the enzyme activity is respectively improved by 1.37 times and 1.22 times. The enzyme production ability and catalytic efficiency of the modifiedstrain are improved, and after HPLC (High Performance Liquid Chromatography) detection and purification, the content of chitin oligose in the mutant discovers that the sugar yield of the mutant F273wis 1.65g / L when catalyzed for 3 hours, the content of GlcNAc is 0.12g / L, and the content of (GlcNAc)2 is 1.53g / L. Compared with a wild strain, the chitin enzyme mutant disclosed by the invention has the advantage that the enzyme activity is improved by 49.7%.

Description

technical field [0001] The invention relates to a chitinase mutant with improved enzyme activity, belonging to the technical fields of enzyme engineering and genetic engineering. Background technique [0002] Chitinase (EC 3.2.1.14.), also known as chitinase, can catalyze the breakage of insoluble chitin sugar chain β-1,4 glycosidic bonds to generate water-soluble chitooligosaccharides. Because chitinase can be used as a biological control agent for the development of transgenic plants to achieve disease resistance; it can also be used for protoplast separation, cytochemical localization, and production of single-cell proteins. Therefore, it is widely used in agriculture, biotechnology, food, and health have important applications. The chitosan oligosaccharide, the hydrolyzed product of chitinase, can improve the body's immunity, inhibit the growth of tumor cells, activate and proliferate bifidobacteria in the human intestinal tract, antibacterial, antiseptic, and moisturiz...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/75C12N1/21C12R1/125
CPCC12N9/2442C12N15/75C12Y302/01014
Inventor 刘龙潘梦妍吕雪芹堵国成李江华陈坚
Owner JIANGNAN UNIV
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