Mannanase pman5a mutant with improved thermotolerance and its gene and application

A mannanase and mutant technology, applied in the field of agricultural biology, can solve the problems of low catalytic activity, poor β-mannanase tolerance and the like

Active Publication Date: 2021-03-26
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of poor tolerance and low catalytic activity of existing β-mannanases, the present invention mutates the amino acids H, F, L and A at positions 93, 94, 356 and 389 in PMan5A to Y, Y, H, and P, or combined mutations, to obtain mannanase mutants with improved thermostability

Method used

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  • Mannanase pman5a mutant with improved thermotolerance and its gene and application
  • Mannanase pman5a mutant with improved thermotolerance and its gene and application
  • Mannanase pman5a mutant with improved thermotolerance and its gene and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 Construction of mannanase mutant engineering bacteria

[0064] (1) Construction of expression vector and expression in Pichia pastoris

[0065] Using the GH5 family mannanase Pman5A-pPIC9 derived from Penicillium sp.WN1 as a template, mutant primers H93Y F / R, F94Y F / R, L356H F / R and For A389P F / R (as shown in Table 1), the point mutation kit was used to perform PCR amplification first, then the PCR product was demethylated by DMTase and transformed into DMT competent cells, and finally the single clone was selected for verification and positive selection The transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of recombinant plasmids.

[0066] Table 1 takes wild mannanase PMan5A as the primers used to construct mutants

[0067]

[0068] Linearize the recombinant vector with the correct sequencing of the expression vector pPIC9 with endonuclease DraI and transform it into P...

Embodiment 2

[0071] Embodiment 2 Mannanase mutants and the preparation of wild enzyme enzyme liquid

[0072] (1) Expression of mutant genes at shake flask level in Pichia pastoris

[0073] Pick the transformant with the highest enzyme activity, inoculate it in 30mL YPD medium and culture it for 48h for seed scale-up cultivation, then inoculate it into a 1L Erlenmeyer flask with 300mL BMGY medium according to the inoculation volume of 1%, and culture it on a shaker at 30°C and 220rpm After 48 hours, the culture solution was centrifuged at 3000 g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 200 mL of BMMY medium containing 0.5% methanol, and placed again at 30°C and 220 rpm to induce culture. Add 1 mL of methanol every 12 hours to keep the concentration of methanol in the bacterial solution at 0.5%, and take the supernatant for enzyme activity detection.

[0074] (2) Purification of mutant mannanase

[0075] Collect the supernatant of the mutant mannanase...

Embodiment 3

[0076] Example 3 Activity analysis and property determination of mannanase mutants and wild enzyme Pman5A

[0077] Adopt DNS method to carry out activity analysis to mannanase of the present invention, concrete method is as follows:

[0078] Under the given pH and temperature conditions, 1 mL of the reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, reacted for 10 min, added 1.5 mL of DNS to terminate the reaction, and boiled for 5 min. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose carob gum to produce 1 μmol reducing sugar per minute under given conditions.

[0079] (1) Determination of optimum temperature and temperature stability of mutant and wild enzyme Pman5A

[0080] The enzyme-catalyzed reaction was carried out in a pH5.0 citric acid-disodium hydrogen phosphate buffer system and at different temperatures (40-90° C.), and the optimum te...

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Abstract

The invention discloses a mannanase mutant with improved heat resistance and its gene and application, which uses the GH5 family mannanase PMan5A derived from Penicillium sp.WN1 as the female parent, and adopts molecular biology means The histidine (H), phenylalanine (F), leucine (L) and alanine (A) at positions 93, 94, 356 and 389 in the female parent were mutated into tyrosine ( Y), tyrosine (Y), histidine (H) and proline (P). The results showed that the heat resistance of the single point mutants H93Y, L356H and A389P were significantly improved compared with the wild enzyme PMan5A, and the combined mutants H93Y / F94Y, H93Y / L356H, H93Y / A389P, L356H / A389P, H93Y / F94Y / The thermotolerance of L356H, H93Y / L356H / A389P, H93Y / F94Y / L356H / A389P showed the additive effect of single point mutations, indicating that the 93, 94, 356 and 389 sites of PMan5A were involved in the thermotolerance of GH5 family mannanases This may have important guiding significance for the research on the mechanism of thermotolerance of other GH5 family (α / β)8 barrel enzymes.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology. Specifically, the present invention relates to mannanase PMan5A mutants with improved thermostability, genes and applications thereof. Background technique [0002] Mannan is the main component of plant hemicellulose, which mainly exists in cork plants and some special structures such as plant seeds. It is also an important plant feed material. The structure of mannan is relatively complex. Its main chain is a linear polysaccharide connected by 1,4-β-D-mannopyranoside bonds, and the main chain or side chain has various substituents. Due to the diversity and structure complexity of mannan, its complete hydrolysis requires the synergistic action of multiple enzymes, including endo-β-mannanase, exo-β-mannosidase, β-glucose glycosidase, acetylmannan esterase and α-galactosidase, etc. Among them, endo-β-mannanase can degrade the β-1,4-glycosidic bond of mannan main chain, and is the enzyme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/81C12R1/84
CPCC12N9/2494C12N15/815C12Y302/01078C12N9/2491C12Y302/01025C07K1/14C12N15/63
Inventor 罗会颖姚斌刘伟娜顾原涂涛王苑王亚茹黄火清柏映国苏小运孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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