A method for increasing the expression of chitinase

A chitin enzyme and expression technology, which is applied in the fields of enzyme engineering and microbial engineering, can solve problems such as complex operation, loss of enzyme activity, and difficulty in screening, and achieve great application prospects and high-efficiency secretion and expression

Active Publication Date: 2020-08-04
JIANGNAN UNIV
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Problems solved by technology

[0004] At present, the microorganisms that have been found to produce chitinase mainly include Paenibacillus barengoltzii, Marine Bacterium (Alteromonas sp.Strain 0-7), Streptomycesthermoviolaceus OPC-520 and Bacillus cereus, etc. However, the chitinase production of these wild strains is very low , only 0.83-1.13U / mL
[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 Modifying the substrate-binding domain and catalytic domain of chitinase to improve the enzyme activity has the uncertainty of random mutation, therefore, it will lead to screening difficulties, 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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  • A method for increasing the expression of chitinase
  • A method for increasing the expression of chitinase
  • A method for increasing the expression of chitinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: Construction of recombinant bacteria

[0048] Specific steps are as follows:

[0049] (1) Using Bacillus subtilis 168 as a template, respectively using NprB-F, NprB-R, AmyE-F, AmyE-R, AprE-F, AprE-R, Bpr-F, Bpr-R, BglS-F, BglS -R, Epr-F, Epr-R, LipA-F, LipA-R, Vpr-F, Vpr-R, YclQ-F, YclQ-R, YweA-F, YweA-R as forward and reverse primers (see table 1-2), 10 signal peptide fragments were amplified by PCR: NprB, AmyE, AprE, BglS, Bpr, Epr, LipA, Vpr, YweA, YclQ; the PCR reaction conditions were: 98°C for 3min, 30 cycles (98 ℃30s, 55℃30s, 72℃30s), 72℃5min;

[0050] (2) Use pP43NMK-chisb (the chisb gene was synthesized by Wuxi Tianlin Biological Co., Ltd. and provided the constructed plasmid) as a template, and p43-F and p43-R as forward and reverse primers (see Table 1-2 ), the linearized carrier fragment containing the chitinase gene whose N-terminus is deleted from its own signal peptide gene was amplified by PCR of the whole plasmid; 72℃4min30s), 72℃5min;...

Embodiment 2

[0065] Embodiment 2: the verification of high-yielding chitinase recombinant bacteria

[0066] Specific steps are as follows:

[0067] The recombinant plasmids pP43NMK-NprB, pP43NMK-AmyE, pP43NMK-AprE, pP43NMK-BglS, pP43NMK-Bpr, pP43NMK-Epr, pP43NMK-LipA, pP43NMK-Vpr, pP43NMK-LipA, pP43NMK-Vpr, pP43NMK-YweA and pP43NMK-YclQ were respectively transformed into Bacillus subtilis WB600, and the selected transformants were inoculated into LB liquid medium, cultured at 37°C for 8 hours, then transferred to TB medium with an inoculum size of 2%, cultured for 12 hours, collected and fermented The supernatant of the fermentation liquid was used to detect the enzyme activity of the fermentation supernatant; at the same time, the cell wall was broken by ultrasonic disruption, and the enzyme activity in the cell was detected.

[0068] The result is as figure 1 Shown (with empty plasmid pP43NMK, recombinant plasmid pP43NMK-chisb, recombinant plasmid pP43NMK-chisb-sp as control).

[0069...

Embodiment 3

[0070] Example 3: Protein electrophoresis verification of chitinase production strains expressing different signal peptides

[0071] The fermentation supernatant in Example 2 was taken, and the recombinant strains containing different signal peptides were subjected to protein sample treatment. System: 30 μL fermentation supernatant, 10 μL 4× protein loading buffer, 99 ° C, 10 min, and then protein electrophoresis. Through dyeing, decolorization and other processes, the results are as follows figure 2 shown.

[0072] The results showed that the bands of the recombinant strains fused with different signal peptides were all obviously thicker, indicating that the yield of chitinase was significantly increased. At the same time, it was found that the recombinant strain pP43NMK-YclQ fused with the YclQ signal peptide had no protein band display, indicating that it had no protein bands. The ability to secrete chitinase extracellularly. The recombinant strain pP43NMK-NprB protein b...

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Abstract

The invention discloses a method for improving expression quantities of chitin enzymes, and belongs to the technical field of enzyme engineering and microorganism engineering. According to the method,genes of signal peptides NprB, AmyE, AprE, BglS, Bpr, Epr, LipA, Vpr, YweA or YclQ are fused to an N end of a chitin enzyme gene, a signal peptide gene of which is excised; and the chitin enzyme genefused with an exogenous peptide gene is expressed in an expression host so as to improve an expression quantity of the chitin enzyme. By fermenting recombinant bacteria obtained by utilizing the method for 12 hours, enzyme activity of chitin enzymes in fermented supernate can be improved to 20.62 U / mL (extracellular enzyme activity) and is about 15 times of wild strain fermentation.

Description

technical field [0001] The invention relates to a method for increasing the expression level of chitinase, which belongs to the technical field of enzyme engineering and microbial 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. [0003] Because chitinase can be used as a biological control agent, it can also be used for protoplast separation, cytochemical localization and production of single-cell protein, so it has very important applications in agriculture and biotechnology; because the hydrolyzate of chitinase is almost Tetraoligosaccharides can improve the body's immunity, inhibit the growth of tumor cells, activate and proliferate bifidobacteria in the human intestine, antibacterial, antiseptic, and moisturizing. Therefore, it also has broad application prospects in industries such as medicine, fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/74C12N9/24C12N1/21
CPCC07K2319/02C12N9/2442C12N15/74C12Y302/01014
Inventor 刘龙潘梦妍吕雪芹堵国成李江华陈坚
Owner JIANGNAN UNIV
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