Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Chitinase low temperature resistant mutant and application thereof

A technology of chitinase and chitin, which is applied in the direction of application, enzyme, and introduction of foreign genetic material by using vectors, etc., can solve the problem of not many low-temperature chitinases, etc.

Active Publication Date: 2019-06-28
DALIAN UNIVERSITY
View PDF4 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there are not many low-temperature chitinases reported

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Chitinase low temperature resistant mutant and application thereof
  • Chitinase low temperature resistant mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023]The gene cloning of embodiment 1 wild-type chitinase

[0024] The inventors have discovered that a chitinase screened from the amino acid sequence of Pseudoalteromonas sp. as shown in SEQ ID NO: 1 is disclosed in the prior art, and its encoded nucleotide is shown in SEQ ID NO: 1. ID NO: 2, expressed and prepared in Escherichia coli. In short, the gene was entrusted to Shanghai Jierui Biotechnology Co., Ltd. to artificially synthesize, then ligated into pET-28(+) vector (New England Biolabs) between the restriction sites of EcoR I and HindIII, and transformed into the large intestine Bacillus 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

[0025] Embodiment 2 mutation screening

[0026] In order to further improve the low temperature 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 respectively for error-prone PCR, carried out multi-point random mutations on the target gene, and found that some mutations had no effect on its low temperature resistance, some mutations even made the low temperature resistance worse, and some mutations, although they could improve heat resistance, were not affected by the mutation. The scope of application of pH narrowed, and none of them met the requirements. In the end, the applicant obtained a combination of mutation sites that can not only significantly improve the low temperature performance, but also expand the scope of application under acidic conditions: R501L, Y555W, F635P, A673S, E689T; specifically, the Arg muta...

Embodiment 3

[0029] Example 3 Expression Purification

[0030] 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.

[0031] Pick up the positive bacteria with the inoculation needle and inoculate them in 5mL LB medium, culture them at 30°C and 200r / min for 12h, then inoculate them in 400mL LB containing 50ug / mL kanamycin with 2% (V / V) inoculum. culture medium at 30°C, 200r / min for 8h. When the bacterial density OD600 reached 0.7, 0.5 mM IPTG was added to induce expression; 16 ° C 170 rpm shaking culture overnight.

[0032] Bacteria were collected by centrifugation, washed with PBS, sterilized by ultrasonication in ice, and the supernatant was collected by c...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention obtains a mutant having 5 amino acid locus mutation in comparison with a wild type by screening a low temperature resistant chitinase from a marine microorganism disclosed in the prior art through PCR introduction mutation. Through appropriate temperature detection, the most suitable temperature of the mutant is reduced by 5 DEG C compared with the wild type, which is more conduciveto the application of the chitinase to the hydrolysis of chitin.

Description

technical field [0001] The invention belongs to the technical field of gene screening and transformation, and in particular relates to a chitinase enzyme (glucosidase) and its application, in particular to a chitinase mutant obtained by screening a chitinase through a gene mutation method and a chitinase resistant to low temperature Application of mutants 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)...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/04C12P19/12C12P19/00C12R1/19
Inventor 王晓辉迟乃玉
Owner DALIAN UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com