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Beta-mannase heat-resisting mutant, application thereof, recombinant strain and application thereof

A technology of mannanase and mutants, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems that the heat resistance does not meet the industrial requirements and the biological activity is unstable, and achieves the effects of reducing production costs and improving heat resistance

Active Publication Date: 2020-07-03
OCEAN UNIV OF CHINA
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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-resisting mutant, application thereof, recombinant strain and application thereof

Examples

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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.

[0059] 8. Example 4: Verification of high-throughput expression of β-mannanase mutants in Pichia pastoris. Pick the activated transformants in Example 3 and inoculate them in 1 mL of BMGY medium, and place them in a 48-well plate at 30°C , 200rmp shaker culture, 24h after the first addition of 1% methanol of the culture volume to induce the expression of the enzyme, and then induce once every 24h during the culture process. After inducing expression for 96 hours, the culture medium was centrifuged to obtain the supernatant, and th...

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Abstract

The invention relates to a beta-mannase heat-resisting mutant, an application thereof, a recombinant strain and an application thereof, and belongs to the fields of genetic engineering and enzyme engineering. A protein surface charge optimization strategy is adopted, mutation sites are reasonably designed and screened, beta-mannase ManAK from aspergillus candidus is improved, then through a high throughput screening method, forward mutation is sorted, and finally, 23 beta-mannase mutants with improved heat stability are obtained. The heat resistance of the obtained beta-mannase mutant is 1.2-3.5 times of that of original genes, the heat resistance is notably improved, the application of the beta-mannase mutant to business is facilitated, and production cost is reduced.

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): C12N9/24C12N15/56C12N1/19C12R1/84
CPCC12N9/2494C12Y302/01078
Inventor 牟海津刘哲民宁晨
Owner OCEAN UNIV OF CHINA
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