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High-temperature alkaline xylanase XYN11A as well as gene and application thereof

A technology of xylanase and mannanase, applied in the field of genetic engineering, can solve problems such as loss, low expression limit industrial production prospects, and inability to adapt to alkaline environment

Active Publication Date: 2013-10-09
WUHAN SUNHY BIOLOGICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the reported xylanases derived from fungi are stable under acidic conditions, but almost lose their activity when the pH exceeds 10.0, and 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 XYN11A as well as gene and application thereof
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  • High-temperature alkaline xylanase XYN11A as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Cloning of Example 1 Xylanase Encoding Gene Xyn11A

[0050] The xylanase gene xyn11A is derived from Humicola. Extraction of Humicola Genomic DNA:

[0051] After 3 days of cultivation, the mycelium was centrifuged at high speed and taken into a mortar, frozen in liquid nitrogen and ground for 5 minutes, then the grinding solution 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 0.2mL TE, and stored at -20°C for later use.

[0052] Since the whole gene sequence of the Humicola sp.S8 strain...

Embodiment 2

[0057] The preparation of embodiment 2 recombinant xylanase

[0058] The expression vector pPIC9 was subjected to double enzyme digestion (EcoR I+Not I), and at the same time, the amplified xyn11A gene encoding xylanase (without signal peptide) was subjected to double enzyme digestion (EcoR I+Not I), and the xyn11A gene encoding the xylanase was cut out. The polycanase gene fragment is connected with the expression vector pPIC9 to obtain the recombinant plasmid pPIC9-xyn11A containing the xyn11A gene xyn11A and transform the Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / xyn11A.

[0059] The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 72 hours of induction, the supernatant was collected by centrifugation, and the activity of xylanase was determined. ...

Embodiment 3

[0061] The activity analysis of embodiment 3 recombinant xylanase

[0062] The recombinant xylanase produced by the recombinant strain GS115 / XYN11A was purified, and the activity was analyzed by DNS method.

[0063] DNS method: The specific method is as follows: at pH 7.0 and 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 field of gene engineering, and particularly relates to high-temperature alkaline xylanase XYN11A as well as a gene and an application thereof. An amino acid sequence of the xylanase XYN11A is as shown in SEQ ID NO.1 or 2. The invention also provides the gene for coding the high-temperature alkaline xylanase XYN11A, a recombinant vector containing the gene, a recombinant strain containing the gene and an application of the gene, wherein a nucleotide sequence of the gene is as shown in SEQ ID NO.4 or 5. The optimum pH of the xylanase provided by the invention is 7.0, and the xylanase has enzyme activity of above 75% in the pH range from 5.5-8.5 and very good pH stability in the pH range from 4.0-12.0; and the optimum temperature of the xylanase is 60 DEG C. The xylanase XYN11A provided by the invention can be used for effectively degrading various types of xylan, has no degradation effect on celluloses, is a novel enzymic preparation and has enormous potential and commercial value when being applied in papermaking and textile industries.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high-temperature alkaline xylanase XYN11A and its gene and application. Background technique [0002] Natural lignocellulosic biomass mainly includes cellulose, hemicellulose and lignin, and its structure is extremely complex. Among them, cellulose is the main skeleton, and lignin and hemicellulose are dispersed in and around the cellulose. Cellulose is the most widely distributed and most abundant in nature. It is a high molecular polymer without side chains formed by anhydroglucopyranose linked by β-1,4-glycosidic bonds. The content of hemicellulose is second to that of cellulose, which is a heterogeneous polymer formed by five-carbon sugars (xylose and arabinose) and six-carbon sugars (mainly mannose, a small amount of glucose and galactose) and uronic acid, It is mainly composed of xylose connected by β-1,4 glycosidic bonds. Lignin is a complex p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N15/81C12N1/19C12P19/14
Inventor 詹志春张菁
Owner WUHAN SUNHY BIOLOGICAL
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