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Beta-xylosidase and application thereof

A technology of xylosidase and gene, applied in the field of β-xylosidase and its application, to achieve the effect of high enzyme activity, wide application and strong tolerance

Pending Publication Date: 2020-12-29
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing β-xylosidase adapts to pH values ​​in the acidic and neutral ranges, and the temperature tolerance is only below 50°C
This limits the use of β-xylosidase in energy conversion, especially bioalcohol fermentation

Method used

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  • Beta-xylosidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Aspergillus oryzae genome extraction

[0056] Inoculate the Aspergillus oryzae sp.FJ0123 strain on a PDA slope, culture at 30°C for 4 days, prepare a spore suspension with normal saline, and adjust to OD 600 2.0, the spore suspension was put into 50mL PDA liquid medium with glass beads, cultured by shaking at 30°C and 180rpm for 24 hours, centrifuged at 12000rpm to collect the bacteria, and the bacteria were quickly ground in liquid nitrogen until it was ground into a fine white powder. Genomic DNA was extracted with Plant Genomic DNA Extraction Kit according to its instructions.

Embodiment 2

[0057] The PCR amplification of embodiment 2β-xylosidase gene

[0058] According to the sequence information of the β-xylosidase gene (XM001817927.1) predicted on NCBI, primers were designed using Primer Premier 5 of Premier Company, and the primer sequences were as follows:

[0059] Ao-Xyl-F1: 5'-ATGCCTGGTGCAGCGT-3' (SEQ ID NO: 3);

[0060] Ao-Xyl-R1: 5'-CTATTGCGGCGCAATCAACT-3' (SEQ ID NO: 4);

[0061] PCR amplification was performed using the Aspergillus oryzae genomic DNA of Example 1 as a template. The reaction parameters are: denaturation at 95°C for 5 min, denaturation at 94°C for 155 sec, gradient annealing at 55-58°C for 5 sec, extension at 72°C for 3 min, and after 30 cycles of incubation at 72°C for 10 min, a fragment of about 2600 bp was obtained, and agarose gel was performed after amplification Electrophoresis verification, electrophoresis results such as figure 1 As indicated, the fragment was recovered and sent to Borui Biotechnology Co., Ltd. for sequencing....

Embodiment 3

[0063] Example 3 Construction of recombinant cloning plasmid pMD19T-Ao-Xyl

[0064] 1) The β-xylosidase gene amplification product is connected to the cloning vector

[0065] The complete β-xylosidase gene obtained in Example 2 was re-amplified, and the obtained product was recovered and purified and ligated with pMD19-T Simple vector (purchased from Takara Company) to construct a recombinant cloning plasmid.

[0066] The ligation reaction was performed according to the instructions provided by the pMD19-T Simple vector ligation kit. Add the following ingredients in sequence to a 0.2mL PCR tube:

[0067]

[0068] After mixing, centrifuge briefly, and connect at 16°C for 4 hours to obtain the ligation product.

[0069] 2) Preparation of Escherichia coli DH5α chemical transformation competent cells

[0070] ①Use the LB plate medium, pick Escherichia coli (strain preserved in glycerol at -20°C) with an inoculation loop, grade and streak on the plate, and incubate upside dow...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to beta-xylosidase and application thereof. The invention provides a gene for encoding the beta-xylosidase, theamino acid sequence of the beta-xylosidase is shown as SEQ ID NO: 1, and the nucleotide sequence of the gene for encoding the beta-xylosidase is shown as SEQ ID NO: 2. The beta-xylosidase provided bythe invention has acid resistance and thermal stability, has the capability of assisting xylanase to hydrolyze xylan, and is beneficial to use in a biomass conversion process.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a β-xylosidase and its application. Background technique [0002] Pollution from wastes with lignocellulosic compositions from industrial and agricultural activities is a major environmental concern; however, they can be a low-cost renewable starting material, a valuable resource that can be used to produce a variety of chemical products and fuels such as ethanol. To produce ethanol from the polysaccharide fraction of the waste, this lignocellulose needs to be efficiently hydrolyzed into xylose. [0003] Xylose can be converted into ethanol through the fermentation of microorganisms such as yeast and Candida candida, and in the process of beer production, due to the high content of β-glucan and xylan in the raw materials, it is produced A series of problems such as turbidity of wine liquid and blockage of beer membrane will appear in the beer produced, which ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N15/81C12N1/19C12R1/84
CPCC12N9/2402C12N15/815Y02E50/10
Inventor 张永辉汪驰陈培旭肖安风杨秋明陈福泉翁惠芬肖琼
Owner JIMEI UNIV
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