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High temperature resistant xylosidase XynB1, gene encoding the enzyme and uses thereof

A technology of xylosidase and encoding gene, applied in the field of xylosidase, can solve problems such as no literature reports, and achieve the effects of good thermal stability and high 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 the xylosidase gene, there are no reports in the literature to obtain thermostable xylosidase and its encoded gene from Bacillus thermophila

Method used

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  • High temperature resistant xylosidase XynB1, gene encoding the enzyme and uses thereof
  • High temperature resistant xylosidase XynB1, gene encoding the enzyme and uses thereof
  • High temperature resistant xylosidase XynB1, gene encoding the enzyme and uses thereof

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Experimental program
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Effect test

Embodiment 1

[0044] 1. Extraction of total DNA from Bacillus denitrophilus NG80-2 (CGMCC No.1228)

[0045] 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 xylosidase:

[0063] Insert the above-mentioned recombinant bacteria H1749 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...

Embodiment 3

[0065] 1. measure the specific activity of xylosidase XynB1 of the present invention at different temperatures:

[0066] The xylosidase obtained in the above-mentioned embodiment two is carried out the mensuration of optimal reaction temperature, and specific method is: preparation xylosidase XynB1 reaction system (100 μ l) is: add substrate p-nitrophenol xyloside to final concentration 10mM, A certain concentration of xylosidase XynB1 was added to 100 μl with 50 mM citrate-sodium citrate buffer, pH5.6. Mix well and react in a water bath at 25~100°C for 20 minutes. After the reaction is over, add 400ul of 1M Na 2 CO 3 The reaction was terminated and the light absorbance was measured at 405 nm. Using p-nitrophenol as the standard curve, calculate the amount of enzyme required to generate 1 μmol of p-nitrophenol per minute, which is defined as 1 enzyme activity unit. For measurement results, see Figure 4 . It can be seen from the figure that the optimum reaction temperatur...

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Abstract

The invention discloses a recombinant xylosidase XynB1 which is genically encoded and expressed in colon bacillus by expression vector built by xylanase XynB1 obtained through Geobacillus thermodenitrificans NG80-2 genome DNA by means of PCR amplification. The optimal reaction temperature of the xylosidase is 60 DEG C, the optimal reaction PH is 5.6, and the xylosidase has good thermal stability in acid environment. The xylosidase is suitable for decomposing oligomerization xylan to generate xylose.

Description

technical field [0001] The present invention relates to a xylosidase, in particular to the cloning and expression of the xylosidase XynB1 gene in Geobacillus thermodenitrificans (Geobacillus thermodenitrificans) NG80-2, the functional identification and the construction of high-temperature resistant xylosidase and xylosidase highly expressed in Escherichia coli The gene encoding the enzyme and its 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 indu...

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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