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High temperature resistant xylanase XynA2, gene encoding the enzyme and uses thereof

A technology of xylanase and coding gene, which is applied in the field of xylanase, can solve the problems that have not been reported in the literature, and achieve the effect of excellent thermal stability

Inactive Publication Date: 2009-05-13
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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  • High temperature resistant xylanase XynA2, gene encoding the enzyme and uses thereof
  • High temperature resistant xylanase XynA2, gene encoding the enzyme and uses thereof
  • High temperature resistant xylanase XynA2, gene encoding the enzyme and uses thereof

Examples

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Effect test

Embodiment 1

[0047] 1. Extraction of total DNA from Bacillus denitrophilus NG80-2 (CGMCC No.1228)

[0048] 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

[0065] Expression, purification and characterization of recombinant xylanase:

[0066] Insert the above-mentioned recombinant bacteria H1398 monoclonal into 20ml 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 at 1% (v / v) inoculum. Kan's LB medium was cultivated at 37°C and 220rpm until the OD600 was 0.6, then IPTG was added to a final concentration of 0.1mM, and induced at 37°C and 180rpm 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 u...

Embodiment 3

[0068] 1. measure the specific activity when xylanase of the present invention acts on the substrate of different sources:

[0069] 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.

[0070] 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 XO502) were tested for enzyme activity. The specific method is: prepare the xylanase XynA2 reaction system (100 μ l) as follows: add oat xylan, beech xylan, birch xylan substrate to a final concentration...

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Abstract

The invention discloses a recombinant xylanase which is genically encoded and expressed in colon bacillus by expression vector built by xylanase XynA2 obtained through Geobacillus thermodenitrificans NG80-2 genome DNA by means of PCR amplification. The optimal reaction temperature of the xylanase is 70 DEG C, the optimal reaction PH is 9.0, and the xylanase has good thermal stability in alkaline environment. The xylanase is suitable for decomposing xylan in hemicellulose to produce functional oligomerization xylan.

Description

technical field [0001] The invention relates to a xylanase, in particular to the cloning, expression and functional identification of the xylanase XynA2 gene in 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 industrial and agricultural waste can be con...

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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