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A modified signal peptide that guides the secretion and expression of thermostable mannanase, recombinant plasmid, genetically engineered bacteria, production method and application

A technology of mannanase and genetically engineered bacteria, applied in the directions of genetic engineering, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of low initial enzyme activity, difficulty in large-scale industrial production, and no guarantee of biological safety of strains. and other problems, to achieve the effect of increasing secretion expression and reducing cost

Active Publication Date: 2021-09-07
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The production of β-mannanase by traditional microbial fermentation is often limited by low initial enzyme activity and no guarantee of biological safety of strains, making it difficult for large-scale industrial production

Method used

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  • A modified signal peptide that guides the secretion and expression of thermostable mannanase, recombinant plasmid, genetically engineered bacteria, production method and application
  • A modified signal peptide that guides the secretion and expression of thermostable mannanase, recombinant plasmid, genetically engineered bacteria, production method and application
  • A modified signal peptide that guides the secretion and expression of thermostable mannanase, recombinant plasmid, genetically engineered bacteria, production method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the construction of starting strain recombinant plasmid

[0027] (1) Cloning of signal peptide GAS1 gene

[0028] According to the gene sequence SEQ ID NO:2 of the signal peptide GAS1 and the characteristics of the α-factor gene sequence on the expression vector pPICZαA, synthetic primers were designed:

[0029] P1: 5'-GCGC TTCGAA ATGTTTAAAATCTCTGTGC-3' (SEQ ID NO: 8)

[0030] P2: 5'-CCGG GAATTC TGCCAAGACTGAACTCAA-3' (SEQ ID NO: 9)

[0031] The underlined part of the primer P1 is the BspT104I restriction site, and the underlined part of the primer P2 is the EcoRI restriction site. Using the Pichia pastoris X33 genome as a template and P1 and P2 as primers, the gene fragment of the signal peptide GAS1 was amplified by PCR.

[0032] (2) Construction of recombinant plasmid containing signal peptide GAS1 gene

[0033] The gene fragment of signal peptide GAS1 obtained by PCR amplification was double-digested with BspT104I and EcoRI, and the expression v...

Embodiment 2

[0041]Example 2: Construction of the starting strain producing thermostable mannanase

[0042] After linearizing the recombinant plasmid pPICZA-GAS1-ManA of the starting strain with restriction endonuclease SacⅠ, it was transformed into Pichia pastoris X33 competent cells by electroporation, and spread on a YPD plate containing 100 μg / mL Zeocin antibiotic Screening was carried out, and the transformants were picked and cultured overnight in YPD liquid medium, and the bacterial liquid PCR was identified, and P1 / P2 and P3 / P4 were used as primers for PCR amplification. The results showed that the gene sequence of the signal peptide GAS1 and heat resistance The gene sequences of mannanase ManA were all integrated into the X33 genome, and the obtained strain producing thermostable mannanase was named X33 / GAS1-ManA.

Embodiment 3

[0043] Example 3: Construction of recombinant plasmid containing modified signal peptide

[0044] (1) Cloning of transformed signal peptide GAS1-3 and GAS1-2K genes

[0045] According to the gene sequence SEQ ID NO:4 and SEQ ID NO:6 of the transformed signal peptide GAS1-3 and GAS1-2K and the characteristics of the α-factor gene sequence on the expression vector pPICZαA, synthetic primers were designed respectively:

[0046] P5: 5'-CGCG TTCGAA ATGTTTAGAAAATCTCTGTGC-3' (SEQ ID NO: 12)

[0047] P2: 5'-CCGG GAATTC TGCCAAGACTGAACTCAA-3' (SEQ ID NO: 9)

[0048] P1: 5'-GCGC TTCGAA ATGTTTAAAATCTCTGTGC-3' (SEQ ID NO: 8)

[0049] P6: 5'-CTCT GAATTC TGCCTTCAAGACTGAACTC-3' (SEQ ID NO: 13)

[0050] The underlined parts of primers P5 and P1 are BspT104I restriction sites, and the underlined parts of primers P2 and P6 are EcoRI restriction sites. Using pPICZA-GAS1-ManA as a template and using P5 / P2 and P1 / P6 as primers respectively, the gene fragments of the modified signal pept...

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Abstract

The invention relates to the field of biology, in particular to a modified signal peptide for guiding the secretion and expression of thermostable mannanase, a recombinant plasmid, a genetically engineered bacterium, a production method and an application. The present invention utilizes a modified signal peptide to guide the secretion and expression of thermostable mannanase in host cells, increases the secretion and expression of thermostable mannanase, and provides corresponding modified signal peptides, recombinant plasmids, genetically engineered bacteria, and thermostable mannanases. Mannanase production method and application of genetically engineered bacteria.

Description

technical field [0001] The invention relates to the biological field, in particular to a modified signal peptide for guiding the secretion and expression of thermostable mannanase, a recombinant plasmid, a genetically engineered bacterium, a production method and an application. Background technique [0002] Mannan is a kind of hemicellulose polysaccharide, which is the second largest component of hemicellulose in nature, and widely exists in plant cell walls and some legume seeds. However, mannan present in plant cell walls or seeds is very easy to swell when exposed to water, its viscosity is greatly increased, it is difficult to hydrolyze, and it is difficult to be directly used by animals and humans, so it will have a very negative impact on the development and utilization of these substances . [0003] β-mannanase is the key enzyme in the process of mannan degradation, which mainly catalyzes the hydrolysis of β-1,4-D-mannose sugar bonds in mannan. The synergistic acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C12N15/56C12N15/81C12N1/19C12N9/24A23K10/18C12R1/84
CPCA23K10/18C07K7/08C12N9/2488C12N15/815C12Y302/01025
Inventor 张莉邓香连熊海容姚银闵琪谭玉青
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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