A heat-resistant xylanase xynb with high enzyme activity at low temperature and its mutant gene, application and gene sequence preparation method

A xylanase and mutant technology, applied in the fields of protein engineering and genetic engineering, can solve the problems of increased production cost, unfavorable product quality, impact, etc., and achieve the effect of less enzyme inactivation and high enzyme activity

Active Publication Date: 2021-10-19
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to prevent this heat inactivation process, it is necessary to add some chemical substances and other methods to protect the enzyme, which not only increases the production cost but also has an adverse effect on the product quality

Method used

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  • A heat-resistant xylanase xynb with high enzyme activity at low temperature and its mutant gene, application and gene sequence preparation method
  • A heat-resistant xylanase xynb with high enzyme activity at low temperature and its mutant gene, application and gene sequence preparation method
  • A heat-resistant xylanase xynb with high enzyme activity at low temperature and its mutant gene, application and gene sequence preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Obtaining of XYNB gene sequence of xylanase mutant strain

[0036] According to sequence comparison, the amino acid sequence of xylanase 1VBR from Thermotoga maritima MSB8 published in PDB and the predicted xylanase (EHA58720.1 ) have an amino acid sequence similarity of 100%. The predicted xylanase gene sequence is a gene sequence (873,589→874,572 bp) in the genome sequence of Thermotoga maritima MSB8.

[0037] Based on the codon usage preference of Pichia pastoris, the rare codons were converted to high-frequency expression codons, and the predicted xylanase (EHA58720.1) gene sequence was compared with the amino acid sequence of xylanase 1VBR Codon optimization was performed to obtain the optimized xylanase 1 VBR gene sequence (NCBI gene number: KR078269). Using the pET-22b(+)-1VBR plasmid as a template, use Taq enzyme and set the concentrations of Mg2+ and Mn2+ in the PCR system to 2.5mM and 0.8mM respectively, and the primer sequences are 5'-CGCCATGG...

Embodiment 2

[0038] Example 2: Construction of recombinant expression plasmid pPIC9K-XYNB containing xylanase mutant strain XYNB gene

[0039] Add the preferred terminator sequence of Pichia pastoris at the 3' end of the xylanase XYNB gene, and introduce restriction endonuclease EcoR I and Not I sites at the 5' end and 3' end respectively, and cross the gene sequence with Wuhan Qingke Innovation Biotechnology Co., Ltd. completed the whole gene synthesis. The optimized xylanase XYNB gene and the secreted expression vector pPIC9K were double-digested with restriction endonucleases EcoR I and Not I, and then connected with ligase to construct the recombinant expression plasmid pPIC9K-XYNB. The recombinant expression plasmid was transformed into Escherichia coli DH5α competent cells, and the positive clone strain pPIC9K-XYNB-DH5α was screened out by PCR verification method.

Embodiment 3

[0040] Example 3: Construction of Pichia pastoris genetically engineered strains that efficiently secrete and express xylanase XYNB

[0041] The LB liquid medium was used to activate the cultured strain pPIC9K-XYNB-DH5α, and the recombinant plasmid pPIC9K-XYNB was extracted. The recombinant plasmid was linearized with restriction endonuclease Bgl II, and the digested product was recovered. Refer to EasySelectTM PichiaExpression Kit to prepare Pichia GS115 competent cells. Take about 10 μg of linearized plasmid and 80 μL of competent cells, mix gently, place on ice for 15 min, transfer to a pre-cooled 0.2 cm electroporation cuvette, add 1 mL of pre-cooled 1 mol / L sorbitol immediately after the 1500V electric shock, and Let it stand in a 30°C incubator for 1 hour, spread it on an MD plate, and incubate it upside down at 30°C for about 48 hours until transformants appear.

[0042] Pick a single colony and inoculate them on MD plates containing 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0 m...

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Abstract

The invention belongs to the field of protein engineering and genetic engineering, and specifically relates to a heat-resistant xylanase XYNB with high enzyme activity at low temperature and its mutant gene, application and gene sequence preparation method. The invention provides a xylanase with high enzyme activity at low temperature Xylanase XYNB has significantly higher enzyme activity than the original enzyme at temperatures below 80°C, while retaining the heat resistance of the original enzyme. It is suitable for industrial fields such as feed additives, food, brewing, and medicine.

Description

technical field [0001] The invention belongs to the field of protein engineering and genetic engineering, and specifically relates to a heat-resistant xylanase XYNB with high enzyme activity at low temperature and its mutant gene, application and gene sequence preparation method. Background technique [0002] Xylan is the most important hemicellulose in plant cell walls, accounting for about 35% of the dry weight of plant cells, and is the most abundant polysaccharide in nature except cellulose. Xylan is a type of hybrid polysaccharide composed of the main chain and some side chain groups of xylose polymerized through β-1,4-glycosidic bonds. Under the action of glycanase, it can be degraded into xylooligosaccharides and xylose which are urgently needed in the international market. However, a large part of xylanase in nature has not been effectively utilized, resulting in a great waste of this resource. [0003] Microbial xylanase (EC 3.2.1.8) is an important industrial enz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N15/81C12N15/10C12N1/21C12N1/19C12R1/19C12R1/84
CPCC12N9/248C12N15/70C12N15/815
Inventor 熊海容王晶
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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