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High temperature alkaline xylanase XYN10A, gene thereof and application thereof

A technology of xylanase and mannanase, which is applied in the field of genetic engineering, can solve the problems of inability to adapt to alkaline environment, loss, low expression amount and limit the prospect of industrial production, and achieves excellent pH stability, high activity, enzyme live stable effect

Active Publication Date: 2012-03-28
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the reported fungal xylanases are stable under acidic pH conditions, and almost lose their activity when the pH exceeds 10.0, which cannot adapt to the alkaline environment in the papermaking and textile industries
In addition, although xylanase produced by bacteria can be active under alkaline conditions, its low expression level limits its industrial production prospects

Method used

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  • High temperature alkaline xylanase XYN10A, gene thereof and application thereof
  • High temperature alkaline xylanase XYN10A, gene thereof and application thereof
  • High temperature alkaline xylanase XYN10A, gene thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Cloning of Example 1 Xylanase Encoding Gene Xyn10A

[0103] Extraction of Humicola sp.S8 genomic DNA:

[0104] After 3 days of culture, the mycelium was centrifuged at high speed and taken into a mortar, frozen in liquid nitrogen and ground for 5 minutes, then the grinding liquid was placed in a 50mL centrifuge tube, 2mL of CTAB extract was added, and lysed in a water bath at 70°C for 2h, every 10min Mix once and centrifuge at 12000rpm for 10min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved in 0.2 mL TE, and stored at -20°C for later use.

[0105] The degenerate primers P1 and P2 were designed and synthesized according to the conserved (WDVVNE and NDY(F)N...

Embodiment 2

[0113] The preparation of embodiment 2 recombinant xylanase

[0114] The expression vector pPIC9 is subjected to double enzyme digestion (SpeI+NotI), and at the same time, the amplified xyn10A gene encoding xylanase (without signal peptide) is subjected to double enzyme digestion (EcoR I+Not I), and the xylan encoding xylan is excised The gene fragment of the enzyme was connected with the expression vector pPIC9 to obtain the recombinant plasmid pPIC9-xyn10A containing the xylanase gene xyn10A and transform Pichia GS115 to obtain the recombinant Pichia strain GS115 / xyn10A; The cDNA of xylanase XYN10A was inserted into the expression vector pPIC9 from which the α-factor signal peptide sequence had been removed by enzyme digestion and ligation, and the recombinant plasmid pPIC-xyn10A-1 containing the gene xyn10A encoding xylanase sequence containing the signal peptide sequence was obtained And transform Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain GS115 / xyn...

Embodiment 3

[0116] The activity analysis of embodiment 3 recombinant xylanase

[0117] The recombinant xylanase produced by recombinant strains GS115 / XYN10A and GS115 / XYN10A-1 was purified, and the activity was analyzed by DNS method.

[0118] DNS method: The specific method is as follows: at pH 7.0, 60°C, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, reacted for 10 minutes, added 1.5 mL of DNS to terminate the reaction, and boiled for 5 minutes. After cooling, the OD value was measured at 540 nm. One enzyme activity unit (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute under given conditions.

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Abstract

The invention relates to the genetic engineering field, and concretely relates to high temperature alkaline xylanase XYN10A, a gene thereof and an application thereof. The new high temperature alkaline xylanase XYN10A provided by the invention has an amino acid sequence represented by SEQ ID NO.1 or 2. The gene which codes the high temperature alkaline xylanase XYN10A and has a nucleotide sequence represented by SEQ ID NO.4 or 5, a recombinant vector containing the gene, a recombinant strain containing the gene, and the application of the xylanase XYN10A are also provided. The xylanase provided by the invention, which has an optimum pH value of 7.0, has an enzymatic activity of more than 75% in the pH value range of 5.0-10.0, and has an enzymatic activity of more than 50% and a good pH stability when the pH value is 12.0, has an optimum temperature of 70DEG C. The xylanase XYN10A of the invention can effectively degrade various types of xylans, cannot degrade celluloses, and performs large potentials in the papermaking industry and the textile industry.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high-temperature alkaline xylanase XYN10A and its gene and application. Background technique [0002] Lignocellulose is mainly a polymer compound composed of three major types of polysaccharides: cellulose, hemicellulose and lignin. Among them, cellulose is the main skeleton, and lignin and hemicellulose are dispersed in the cellulose and its Around, they are connected by covalent bonds, and there is also a certain degree of chemical bonds (Martinez AT, Ruiz-DuenasFJ, Martinez MJ, et al. Enzymatic deligification of plant cell wall: from nature to mill. Curr Opin Biotechnol, 2009, 20:348-357.). Hemicellulose accounts for 20-30%, and is a polysaccharide formed by linear or branched chain polymerization of D-xylose, D-mannose, L-arabinose or D-galactose. In nature, xylan is the main component of hemicellulose and is the most abundant type of hemicellulos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N15/81C12N1/21C12N1/19D21C9/10D06M16/00C12R1/84C12R1/80
Inventor 詹志春张菁
Owner WUHAN SUNHY BIOLOGICAL
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