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Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof

A technology of xylanase and encoding gene, applied in the field of xylanase, can solve problems such as no literature reports, and achieve the effects of good thermal stability and high thermal stability

Inactive Publication Date: 2009-04-08
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many patent reports on xylanase gene, there is no literature report on obtaining thermostable xylanase and its encoded gene from Bacillus thermophila

Method used

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  • Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof
  • Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof
  • Fire resistant xylanase XynA1, gene for encoding the enzyme and uses thereof

Examples

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Effect test

Embodiment 1

[0045] 1. Extraction of total DNA from Bacillus denitrophilus NG80-2 (CGMCC No.1228)

[0046] The extraction of total DNA from Geobacillus thermodenitrificans NG80-2 (CGMCC No.1228) proved that the gene encoding alcohol dehydrogenase can be isolated from the genome of Geobacillus thermodenitrificans NG80-2. Therefore, in this embodiment, the thermophilic denitrophilic Bacillus NG80-2 obtained from the oil well formation water separation of Guan 69-8 block, Dagang Oilfield, Tianjin, China (it is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee) is used. The number is CGMCC No.1228, and the preservation date is October 9, 2004. It has applied for a domestic invention patent and obtained authorization. 3ml of the fresh culture cultured overnight was collected by centrifugation, and the bacteria were suspended in 250μl 50mM Tris buffer (pH8.0), and 10μl 0.4M EDTA (pH8.0) was added, mixed well, incubated at 37℃ for 20min...

Embodiment 2

[0062] Expression, purification and characterization of recombinant xylanase:

[0063] Insert the above-mentioned recombinant bacteria H1739 monoclonal into 20ml of LB medium containing 50μg / ml Kan, culture at 37°C and 180rpm for 12 hours, and then insert the culture into 200ml containing 50μg / ml Kan's LB medium, 37 ° C, 220 rpm to OD 600 When it is 0.6, add IPTG to a final concentration of 0.05mM, and induce at 45°C for 3 hours. The cells were collected by centrifugation at 5000rpm for 5min, suspended in 50mM Tris-HCl (pH8.0) buffer, disrupted by ultrasonic waves, centrifuged at 14000g for 20min, and the supernatant was the crude extract of alcohol dehydrogenase. This supernatant was purified by chelating agarose gel (Chelating Sepharose) nickel affinity column chromatography, and the enzyme preparation obtained showed a band on SDS-PAGE (see image 3 ). The basic properties of this enzyme system were determined using known standard methods of protein chemistry. The molec...

Embodiment 3

[0065] 1. Measure the specific activity of xylanase of the present invention when it acts on xylan substrates from different sources:

[0066] When xylanase is added to animal feed or paper pulp, the impact of its activity and specificity on xylan from different sources should be considered. Rye) xylan model substrates, beech xylan and birch xylan were used as wood-derived xylan model substrates to investigate their specific activities to xylans from different sources.

[0067] The xylanase prepared in the above-mentioned Example 2 can degrade xylan from different sources. This experiment is for three kinds of xylan substrates currently available for purchase: oat xylan (Sigma product number is XO627), beech wood Glycans (Sigma product number X4252) and birch xylan (Sigma product number X0502) were tested for enzyme activity. The specific method is: prepare the xylanase XynA1 reaction system (100 μl) as follows: add oat xylan, beech xylan, birch xylan substrate to a final con...

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Abstract

The invention discloses a xylanase XynA1 which is obtained by the PCR amplification of Geobacillus thermodenitrificans NG80-2 genome DNA and a recombinant xylanase for constructing an expression vector to express the genetic code in Escherichia coil. The optimal reaction temperature of the xylanase is 70 DEG C, the optimum reaction pH is 7.6, and the thermal stability is good in neutral and weak alkaline environments. The xylanase is applicable to decomposing xylan in hemicellulose to produce functional xylooligosaccharides.

Description

technical field [0001] The present invention relates to a xylanase, in particular to the cloning, expression and functional identification of the xylanase XynA1 gene in Geobacillus thermodenitrificans (Geobacillus thermodenitrificans) NG80-2 and the construction of a high-temperature-resistant xylanase highly expressed in Escherichia coli Carbohydrase, gene encoding the enzyme and application. Background technique [0002] With the rapid development of today's society, population growth and resource consumption make the development and utilization of renewable resources a common concern of the international community and academia. Xylan is the main component of hemicellulose, which is widely distributed in the cell walls of higher plants. It is a huge renewable biological resource in nature, and it is also the easiest type of hemicellulose to extract, degrade and utilize. Xylanase and β-xylosidase are the main enzymes that degrade xylan. In the energy industry, xylan in in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N1/21C12R1/19
Inventor 王磊刘雪倩冯露
Owner NANKAI UNIV
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