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Optimized extremely-thermostable xylanase XYNH coding gene and application thereof

A technology of xylanase and coding genes, which is applied in the field of genetic engineering, can solve problems such as inability to complete enzyme digestion and connection, and achieve the effect of reducing production costs

Inactive Publication Date: 2015-04-22
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The original 1VBR gene contains multiple EcoR I and Bgl II restriction sites. When genetic engineering technology is used to construct Pichia pastoris engineering bacteria, the original gene cannot be directly used to complete the restriction enzyme digestion and ligation.

Method used

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  • Optimized extremely-thermostable xylanase XYNH coding gene and application thereof
  • Optimized extremely-thermostable xylanase XYNH coding gene and application thereof
  • Optimized extremely-thermostable xylanase XYNH coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the acquisition of the optimized xylanase XYNH gene sequence

[0042] According to the sequence comparison, the amino acid sequence of xylanase 1VBR from Thermotoga maritima MSB8 published in PDB (SEQ ID NO.2) and the genome sequence of Thermotoga maritima MSB8 (CP007013.1: 1,869,644bp) predicted xylanase The amino acid sequence similarity of glycanase (EHA58720.1) is 100%. The predicted xylanase gene sequence is a gene sequence (873,589→874,572 bp) in the genome sequence of Thermotoga maritima MSB8.

[0043] 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 XYNH gene sequence.

Embodiment 2

[0044] Embodiment 2: Construction of the recombinant expression plasmid pPIC9K-XYNH containing the optimized xylanase XYNH gene

[0045] The terminator sequence preferred by Pichia pastoris was added to the 3' end of the optimized xylanase XYNH 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.

[0046] The optimized xylanase XYNH gene and the secreted expression vector pPIC9K were double digested with restriction endonucleases EcoR I and Not I, and then the two were connected with ligase to construct the recombinant expression plasmid pPIC9K-XYNH. The recombinant expression plasmid was transformed into Escherichia coli DH5α competent cells, and the positive clone strain pPIC9K-XYNH-DH5α was screened out by PCR verification method.

Embodiment 3

[0047] Example 3: Construction of Pichia pastoris genetic engineering strains that efficiently secrete and express xylanase XYNH

[0048] The LB liquid medium was used to activate the cultured strain pPIC9K-XYNH-DH5α, and the recombinant plasmid pPIC9K-XYNH was extracted. The recombinant plasmid was linearized with restriction endonuclease Bgl II, and the digested product was recovered. Refer to EasySelect TM Pichia Expression 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.

[0049] Pick single colonies and inoculate them on MD plates containing 0.25, 0.5, 1.0, 2.0, 3.0 mg / mL G418...

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Abstract

The invention relates to the field of genetic engineering, in particular to an optimized extremely-thermostable xylanase XYNH coding gene and application thereof. The nucleotide sequence of the nucleotide sequence is shown as SEQ ID NO:1. By means of the optimized extremely-thermostable xylanase XYNH coding gene and application, the problem of expression efficiency reduction caused by the codon preference of extremely-thermostable xylanase genes coming from bacteria in an eukaryotic expression system is solved; and xylanase XYNH which is excellent in character and has not been used for pichia pastoris expressions in the global yet is provided.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an optimized extreme heat-resistant xylanase XYNH coding gene and application thereof. 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-xylosidic bonds inside xylan to generate ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/70C12N15/81C12N9/42
Inventor 熊海容詹志春王亚伟周樱余天意
Owner WUHAN SUNHY BIOLOGICAL
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