High-specific-activity acidic mannase mutant

A technology of mannanase and mutants, which is applied in the field of acidic mannanase mutants, can solve the problems of low mannanase production and inability to meet industrial production, and achieve accelerated wide application, increased specific activity, and reduced production cost effect

Active Publication Date: 2020-05-08
山东康地恩生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of mannanase produced by natural strains

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0051] Example 1 Amplification of wild-type acid mannanase gene

[0052] Take Aspergillus niger ( Aspergillus niger ) The genome is used as a template for PCR amplification. The PCR primers M20-F1 and M20-R1 are as follows:

[0053] M20-F1: GCT GAATTC GGCCTCCAATTCACCATTGATGGCG (underlined is the restriction enzyme EcoRI recognition site)

[0054] M20-R1: CTG GCGGCCGC TTAGGCGCTATCAATAGCAG (underlined is restriction enzyme NotI recognition site)

[0055] The PCR product was recovered by gel, connected to the pEASY-T vector, transformed into E. coli DH5α, and the correct transformants were picked for sequencing. Sequencing results showed that the nucleotide sequence of the amplified gene fragment was SEQ ID NO: 2, and the encoded amino acid sequence was SEQ ID NO: 1. Through NCBI BLAST comparison, it was found that SEQ ID NO: 1 has a sequence similarity of up to 100% with the acid mannanase derived from Aspergillus niger, thus confirming that the gene obtained by PCR is the acid mann...

Example Embodiment

[0056] Example 2 Construction of Pichia pastoris engineered strain expressing wild-type acid mannanase

[0057] The acid mannanase M20 gene described in Example 1 was connected to the expression vector pPIC9K through EcoRI and Not I sites to construct the expression vector pPIC9K-M20.

[0058] The expression vector pPIC9K-M20 was linearized with Sal I, and the linearized fragment of the expression plasmid was transformed into the host cell Pichia pastoris GS115 by electroporation, and the Pichia pastoris recombinant strain GS115 / pPIC9K-M20 was screened on the MD plate, and then it Select multiple copies of transformants on YPD plates with different concentrations of geneticin.

[0059] The selected recombinant positive transformant expressing acid mannanase M20 was named Pichia pastoris M20 ( Pichia pastoris M20), transfer to BMGY medium, 30°C, 250 rpm shaking culture for 1 d; then transfer to BMMY medium, 30°C, 250 rpm shaking culture; add 0.5% methanol every day to induce expressi...

Example Embodiment

[0091] Example 3 Screening of mutants of high specific activity acid mannanase

[0092] In order to further improve the specific activity of acid mannanase M20, the applicant conducted a large number of mutation screenings for the enzyme through directed evolution technology.

[0093] Using the M20 gene as a template, using the primers M20-F1 and M20-R1 described in Example 1, PCR amplification was performed with GeneMorph II Random Mutation PCR Kit (Stratagene), the PCR product was recovered by gel, and EcoRI and Not I were digested After treatment, it was connected with pET21a vector after the same enzyme digestion, transformed into E. coli BL21(DE3), spread on LB+Amp plate, cultivated upside down at 37℃, when transformants appeared, pick them one by one to 96-well plate with toothpicks , Add 150 ul LB+Amp medium containing 0.1mM IPTG to each well, incubate at 37℃ 220 rpm for about 6 hours, centrifuge and discard the supernatant, resuspend the bacteria in buffer, repeatedly freez...

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Abstract

The invention relates to the technical field of protein engineering modification, and particularly provides a high-specific-activity mannase mutant. Compared with a wild type, the mannase mutant provided by the invention has the advantages that the specific activity of the mannase mutant is generally improved by 4.4%-38.97%. The specific activity of the mannase mutant is improved, the production cost of mannase is reduced favorably, wide application of the mannase in the field of feed is accelerated, and the market prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of protein engineering transformation, and in particular relates to an acidic mannanase mutant with improved specific activity. Background technique [0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Mannan is an important component of plant hemicellulose. It is a linear polymer connected by β-1,4-D-mannose. The side chains of the polysaccharide mainly contain glucosyl, acetyl and hemicellulose. Lactosyl and other substituent groups. β-mannanase (β-mannanase EC3.2.1.78) is an endohydrolase that hydrolyzes mannan. It degrades the β-1,4 glycosidic bond of the mannose backbone in an endo-cutting manner and releases a short β -l,4 mannan oligosaccharides. In recent years, with the discovery of the physiological functions of mannan oligosaccharides, the rise of green feed, the enhancement of people's awareness of environmental protection, and the research on the regeneration...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2494C12N15/815C12Y302/01078Y02P60/87
Inventor 吴秀秀宋嵘锡黄亦钧
Owner 山东康地恩生物科技有限公司
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