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

Beta-mannase heat-resistant mutant M20, recombinant strain and application of recombinant bacterium

A technology of mannanase and mutants, which is applied in the field of genetic engineering and enzyme engineering, and can solve problems such as heat resistance not meeting industrial requirements and unstable biological activity

Inactive Publication Date: 2021-09-10
OCEAN UNIV OF CHINA
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the heat resistance of β-mannanase on the market often does not meet the industrial requirements, resulting in unstable biological activity during production and transportation, which seriously limits the application of this enzyme

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
  • Beta-mannase heat-resistant mutant M20, recombinant strain and application of recombinant bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0055] 6. Example 2: Construction of β-mannanase site-directed mutant recombinant vector

[0056] Using the recombinant vector pPICZαA-ManAK containing the ManAK gene as a template, add primers for the mutation site, and perform PCR amplification with high-fidelity enzymes. The product is digested with DpnⅠ and transformed into DH5α E. Positive clones were screened on the LB plate, and the sequencing work was completed by Qingdao Ruibo Xingke Co., Ltd.

Embodiment 3

[0057] 7. Example 3: Construction of β-mannanase site-directed mutant expression vector

[0058] The positive clones with correct sequencing after mutation in Example 2 were extracted and purified, and the corresponding plasmids were linearized with Sac I single enzyme digestion, electrotransformed into Pichia pastoris X33, and screened on MD plates to obtain the transformants of each mutant expression strain. Transfer to YPD plate for activation.

Embodiment 4

[0059] 8. Example 4: Verification of high-throughput expression of β-mannanase mutants in Pichia pastoris

[0060] Pick the activated transformant in Example 3 and inoculate it into 1mL of BMGY medium, culture it in a 48-well plate at 30°C and 200rmp on a shaker, add 1% methanol of the culture volume for the first time after 24h to induce the expression of the enzyme, and then the culture process Induced once every 24h. After inducing expression for 96 hours, the culture medium was centrifuged to obtain the supernatant, and the activity and thermal stability of β-mannanase in the fermentation medium supernatant were determined.

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 relates to a beta-mannase heat-resistant mutant M20, a recombinant strain and application of the recombinant strain, and belongs to the field of gene engineering and enzyme engineering. According to the invention, a protein surface charge optimization strategy is adopted, mutation sites are rationally designed and screened, beta-mannase ManAK from aspergillus candidus is improved, then forward mutation is selected through a high-throughput screening method, and finally, 23 beta-mannase mutants with improved thermal stability are obtained. The heat resistance of the beta-mannase mutant obtained by the invention is 1.2-3.5 times of that of an original gene, and the heat resistance is remarkably improved so as to benefit for implementing commercial application of the beta-mannase mutant, thereby reducing the production cost.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and enzyme engineering, and specifically relates to a kind of beta-mannanase heat-resistant mutants, recombinant bacteria and applications thereof. Background technique [0002] Mannan is the second largest component of hemicellulose, composed of mannopyranose linked by β-1,4 glycosidic bonds. The complete degradation of mannan requires the synergistic action of mannanase, mannosidase, glucosidase, galactosidase and deacetylase. Among them, mannanase is the key enzyme in the degradation process. [0003] β-mannanase (EC 3.2.1.78) is capable of hydrolyzing mannan into mannose and mannooligosaccharides by catalyzing β-1,4 mannosidic linkages. As an important industrial enzyme preparation, the enzyme is widely used in renewable energy fields such as animal feed, food processing, medicine, textile, biological bleaching and petroleum. Some of these application fields, especially feed pelleting, r...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/81C12N1/19C12R1/84
CPCC12N9/2494C12N15/815C12Y302/01078
Inventor 牟海津刘哲民宁晨张芳
Owner OCEAN UNIV OF CHINA
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