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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
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  • Abstract
  • Description
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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

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  • 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.

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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

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Application Information

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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
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