Mutant XYNR of extreme heat-resistant xylanase 1VBR and use thereof

A technology of xylanase and mutants, which is applied in the fields of protein engineering and genetic engineering, can solve the problems of unfavorable product quality, impact, and increased production costs, and achieve the effects of improving stability, good adaptability, and strong stability

Active Publication Date: 2018-05-11
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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  • Mutant XYNR of extreme heat-resistant xylanase 1VBR and use thereof
  • Mutant XYNR of extreme heat-resistant xylanase 1VBR and use thereof
  • Mutant XYNR of extreme heat-resistant xylanase 1VBR and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Obtaining of XYNR gene sequence of xylanase mutant

[0032] 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.

[0033] 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 carried out to obtain the optimized xylanase 1VBR gene sequence. Based on this template, the 290th amino acid in the amino acid sequence of xylanase 1VBR was changed from phenylalanine to valine by gene site-directed mutagenesis to obtain the gene sequence of xylanase mutant XYNR, As shown in SEQ ...

Embodiment 2

[0034] Example 2: Construction of recombinant expression plasmid pPIC9K-XYNR containing xylanase mutant XYNR gene

[0035] The terminator sequence preferred by Pichia pastoris was added to the 3' end of the xylanase mutant XYNR gene, and restriction endonuclease EcoR I and Not I sites were introduced at the 5' end and 3' end respectively, and the gene sequence was Hand over to Wuhan Qingke Innovation Biotechnology Co., Ltd. to complete the whole gene synthesis. The optimized xylanase mutant XYNR 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-XYNR. The recombinant expression plasmid was transformed into Escherichia coli DH5α competent cells, and the positive clone strain pPIC9K-XYNR-DH5α was screened out by PCR verification method.

Embodiment 3

[0036] Example 3: Construction of Pichia pastoris genetically engineered strains that efficiently secrete and express xylanase mutant XYNR

[0037] The LB liquid medium was used to activate the cultured strain pPIC9K-XYNR-DH5α, and the recombinant plasmid pPIC9K-XYNR was extracted. The recombinant plasmid was linearized with restriction endonuclease Bgl II, and the digested product was recovered. Refer to EasySelect TM PichiaExpression Kit prepared 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.

[0038] Pick a single colony and inoculate them on MD plates containing 0.25, 0.5, 1.0, 2.0, 3.0 a...

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Abstract

The invention provides a mutant XYNR of an extreme heat-resistant xylanase 1VBR. The mutant XYNR is prepared through mutation of phenylalanine at the 290th of an amino acid sequence of the xylanase 1VBR into valine and has an amino acid sequence shown in the formula of SEQ ID NO.1. The invention also provides a gene for encoding the mutant XYNR. The gene has a nucleotide sequence shown in the formula of SEQ ID NO.2. The invention also provides a recombinant expression vector pPIC9K-XYNR with the gene for encoding the mutant XYNR and a recombinant strain with the recombinant expression vector.After the amino acid sequence of the xylanase 1VBR is subjected to site-directed mutagenesis, the high heat-resistant mutant XYNR is obtained, is highly expressed in a pichia pastoris expression system and has a wide application prospect in the fields of feed additives, health foods, papermaking, washing, brewing, textiles and pharmacy.

Description

technical field [0001] The invention belongs to the field of protein engineering and genetic engineering, and specifically relates to a mutant XYNR amino acid sequence of an extreme heat-resistant xylanase and its application. 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 enzyme that randomly catalyzes the hydrolysis of β-1,4-D-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12N1/21C12P19/14C12R1/19C12R1/84
CPCC12N9/2482C12P19/14C12Y302/01008
Inventor 熊海容
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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