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An acid-resistant mutant of low temperature-resistant chitinase and its application

A technology of chitinase and chitin, applied in the direction of application, enzyme, hydrolase, etc., can solve problems such as acid sensitivity

Active Publication Date: 2022-02-22
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] So far, the reported acid chitinases are relatively sensitive to acid

Method used

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  • An acid-resistant mutant of low temperature-resistant chitinase and its application
  • An acid-resistant mutant of low temperature-resistant chitinase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The gene cloning of embodiment 1 wild-type chitinase

[0025] The inventors have discovered the acid-resistant chitinase disclosed in the prior art, which is screened from Pseudoalteromonas sp., has an optimum temperature of 35°C, and can maintain 25% activity at 10°C. Belonging to acid-resistant enzymes, the chitinase whose amino acid sequence is shown in SEQ ID NO: 1 and whose coding nucleotide is shown in SEQ ID NO: 2 is expressed and prepared in Escherichia coli. In short, Shanghai Jierui Biotechnology Co., Ltd. was entrusted to artificially synthesize the gene, and then ligated into pET-28(+) vector (New England Biolabs) between the restriction sites of EcoR I and Hind III, and transformed into Escherichia coli DH5α was screened on an LB plate containing 50 mg / L ampicillin. The expression vector contained in the screened positive bacteria was named pET-chi, and the plasmid was extracted and identified correctly by sequencing.

Embodiment 2

[0026] Embodiment 2 mutation screening

[0027] In order to further improve the acid resistance of the above-mentioned chitinase (the amino acid sequence is SEQ ID NO: 1), the applicant used the recombinant plasmid pET-chi constructed in Example 1 as a template to design random mutation primers for error-prone PCR. , the target gene was randomly mutated at multiple points, and it was found that some mutations had no effect on its acid resistance, some mutations even made the acid stability worse, and some mutations, although they could improve acid resistance, had low temperature resistance after mutation. The performance has been reduced and none of them meet the requirements. In the end, the applicant obtained an enzyme that can significantly improve acid resistance and maintain an acid resistance activity equivalent to that of the wild type. The specific mutation combinations are: I861S, A875C, V894P, G903R, H914K, A927N, M942W; specifically the 861st Ile is mutated to Ser...

Embodiment 3

[0030] Example 3 Expression Purification

[0031] Competent cells of Escherichia coli BL21 (DE3) were prepared by methods known in the art, and the wild-type expression vectors constructed in Example 1 and Example 2 and the expression vectors of mutants were respectively transformed into competent cells by the heat shock method, and positive screening results were obtained. The clones were verified by PCR and sequencing for later use.

[0032] Pick the positive bacteria from the inoculation needle and inoculate them in 5mL LB medium, culture at 30°C, 200r / min for 12h, then inoculate 2% (V / V) inoculum in 400mL LB medium, at 30°C, 200r / min / min for 8 hours. When the bacterial density OD600 reached 0.6, 0.5mM IPTG was added to induce expression. In order to prevent the formation of inclusion bodies, the expression condition was low temperature induction at 16°C overnight.

[0033] The bacteria were collected by centrifugation at 4°C, resuspended in PBS, sonicated on ice, and th...

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Abstract

The present application obtained a mutant with 7 amino acid site mutations compared with the wild type by screening the acid-resistant chitinase from marine microorganisms disclosed in the prior art and introducing mutations by PCR. After the most suitable pH It is detected that the most suitable pH value of the mutant is 1.5 lower than that of the wild type, which is more conducive to the application of the enzyme in the hydrolysis of chitin.

Description

technical field [0001] The invention belongs to the technical field of gene screening and modification, in particular to a chitinase enzyme (chitinase) and its application, in particular to a chitinase mutant and chitinase acid-resistant mutation obtained by screening a gene mutation method Application of body in chitin degradation. Background technique [0002] Chitin, also known as chitin and chitin, is widely found in the shells of crustaceans, molluscs and arthropods, such as shrimp, crabs, locusts, etc., and in the cell walls of algae, shellfish, fungi and higher plants. It also exists widely and is the second largest biological resource in nature after cellulose, but it has more special functions than cellulose. Chitin is a white or off-white, translucent flaky crystal, odorless, and is a natural mucopolysaccharide. The chemical name is 1,4-2-acetylamino-2-deoxy-β-D glucan, and the basic unit is acetylglucosamine (GlcNAc), which is a linear polymer. The chemical stru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12R1/19
CPCC12N9/2442C12N15/70C12Y302/01014
Inventor 王晓辉张庆芳
Owner DALIAN UNIV
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