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Super-heat-resistant xylanase XynGold and gene and application

A xylanase and super heat-resistant technology, applied in the field of genetic engineering, can solve the problems of loss of activity, low enzyme specific activity and high production cost, and achieve the effects of increasing expression, increasing enzyme activity and improving activity.

Active Publication Date: 2019-08-13
WUHAN POLYTECHNIC UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although xylanase has wide application prospects in food, feed, papermaking and other fields, there are still key problems to be solved: 1) the specific activity of the enzyme is low
Naturally occurring xylanase activity is generally low, and the hydrolysis efficiency of xylan is low
2) The output of xylanase in industrial production is low, and the production cost is high
3) The high-temperature stability of xylanase is poor, especially in the feed industry, the high-temperature granulation process can easily inactivate heat-labile xylanase

Method used

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  • Super-heat-resistant xylanase XynGold and gene and application
  • Super-heat-resistant xylanase XynGold and gene and application
  • Super-heat-resistant xylanase XynGold and gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example is used to illustrate the site-directed mutagenesis of xylanase gene.

[0039] Based on the xylanase gene xyn (SEQ ID NO: 4), the asparagine at position 68 in the original xylanase Xyn was mutated to glutamine (N68Y) by site-directed mutagenesis technology, and the glutamine at position 96 was mutated Amino acid was mutated to tryptophan (E96W). The xylanase obtained after point mutation (N68Y, E96W) is XynGold (SEQ ID NO: 1). The specific implementation steps include:

[0040] (1) The N68Q mutation was introduced into xylanase Xyn (SEQ ID NO: 2) by overlapping extension PCR technique. Using the xyn10B gene as a template, primers NQF1 (SEQ ID NO: 5, 5'-GAATTCAATGAAAATATTACCTTCTGTG-3') and NQR1 (SEQ ID NO: 6, 5'-GTATCCCACTTCATCTGGTTCTCAGGGGTCAGGATG-3') were used to amplify the first half of the xyn gene nqF1 and primers NQF2 (SEQ ID NO: 7, 5'-GTATCCCACTTCATCTGGTTCTCAGGGGTCAGGATG-3') and NQR2 (SEQ ID NO: 8, 5'-GCGGCCGCCTCATTTTCTTTCTTCTATC-3') were used to a...

Embodiment 2

[0045] This example is used to illustrate the optimization of the codons of the xylanase gene to obtain the gene xyngold.

[0046] In this example, based on the amino acid sequence of the mutated xylanase, the nucleotide sequence of the xylanase gene was artificially redesigned using DNA 2.0 software. Specifically include: compared with the original xylanase gene (SEQ ID NO: 4), high-frequency codons are used to replace low-frequency codons; the complexity and minimum free energy of the secondary structure of the mRNA encoded by the gene are reduced ( figure 2 ). The xyngold sequence of the xylanase gene was designed with the aid of computer, and the xyngold fragment of the xylanase gene was obtained by artificial synthesis.

[0047] In this embodiment, according to the amino acid sequence of xylanase, a brand-new xylanase gene sequence is artificially designed, and the xylanase gene fragment is obtained by artificial synthesis, and its base sequence is as SEQ ID NO: 3 and...

Embodiment 3

[0050] This example is used to illustrate the construction of xyngold recombinant expression strain of xylanase gene.

[0051] (1) Restriction sites EcoR I and Not I were added to both ends of the xyngold xylanase gene, and digested by EcoRI and Not I; similarly, the vector pPICZαA was digested by EcoRI and Not I. Among them, the enzyme digestion system is: 3mg DNA, 10UEcoR I, 10U Not I, 20μL of Buffer H and water to make up to 300μL volume, and enzyme digestion at 37°C for 4h. The result is as Figure 3A As shown (in the figure: M is DL5000 DNA Marker, lane 1 is the pPICZαA recombinant expression vector after enzyme digestion; lane 2 is the xyngold fragment after enzyme digestion; it can be seen from the figure that the sizes of pPICZαA and xyngold fragments are 3.7kb and 1.0kb respectively Left and right, in line with reality.

[0052] (2) Ligate the xyngold fragment of the xylanase gene and the pPICZαA fragment through T4 DNA ligase to obtain the pPIC-xyngold recombinant ...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to a super-heat-resistant xylanase XynGold and gene and application. The core amino acid sequence of the xylanase XynGold is shown as SEQ ID NO: 1, and the xylan base sequence has 95% or above identity with the sequence of SEQ ID NO: 3. The expression level of the artificially synthesized xylanase gene xyngold in pichia pastoris cells is significantly increased, the activity, temperature stability and expression level of the xylanase are significantly improved, and the high-enzyme-activity and high-temperature-resistant xylanase and gene with the high expression level in pichia pastoris are obtained.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to a super heat-resistant xylanase XynGold and its gene sequence, a recombinant expression vector, a recombinant expression strain, the application of the xylanase XynGold and a hydrolyzed wood Glycan approach. Background technique [0002] Xylan is a major component of hemicellulose-like substances present in plant cell walls. It accounts for about 15% to 35% of the dry weight of cells. Xylan is the second largest renewable polysaccharide resource after cellulose in nature. However, xylan can only be utilized by organisms after being hydrolyzed by xylanase into short-chain xylooligosaccharides and xylose and other small molecules. [0003] Xylanases are a class of enzymes capable of hydrolyzing xylan into xylose or xylooligosaccharides. It mainly includes β-1,4-endonuclease, β-1,4-exonuclease and xylosidase, etc. Among them, β-1,4-endonuclease plays...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/248C12N15/815C12P19/14C12P19/02C12P19/04C12P19/12
Inventor 杨江科上官芳韩正刚
Owner WUHAN POLYTECHNIC UNIVERSITY