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High-temperature-resistant xylanase gene and application thereof

A xylanase and gene technology, applied in the field of genetic engineering, can solve the problems of mild reaction conditions and specific reaction, and achieve the effect of good application prospects

Active Publication Date: 2020-07-28
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the physical and chemical methods have their own advantages and disadvantages, but compared with the physical and chemical methods, the enzymatic hydrolysis method has mild reaction conditions and specific reaction.

Method used

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  • High-temperature-resistant xylanase gene and application thereof
  • High-temperature-resistant xylanase gene and application thereof
  • High-temperature-resistant xylanase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The cultivation of embodiment 1 sulfur Aspergillus and the extraction of DNA thereof

[0031] Aspergillus thurophilus CGMCC No.0608 (disclosed in Chinese patent CN01141789.7) was inoculated into sterilized medium (3.5g potato dextrose agar medium, 5g D-maltose, 1g yeast extract, 2g peptone, dissolved in 100mL of distilled water, divided into 50mL Erlenmeyer flasks, then autoclaved for 20min), and cultured on a shaker at 30°C for 72h. After the bacteria were separated, they were ground in liquid nitrogen, and the whole gene of Aspergillus thiocolor was extracted using a fungal DNA extraction kit (Omega, USA), and the whole gene of Aspergillus thiocolor was sequenced.

Embodiment 2

[0032] Embodiment 2 xylanase xyn10A gene cloning

[0033] Analyzing the results of the whole genome sequencing of Aspergillus sulforaphane, a new xylanase gene sequence xyn10A was found, the gene sequence was 1212bp (as shown in SEQ ID NO.1), encoding 404 amino acids (as shown in SEQ ID NO.3) , theoretical molecular weight 43.64kDa. Wherein the N-terminal 22aa is a signal peptide sequence. Using the BLAST online comparison analysis of the NCBI website, it was found that the sequence similarity with the endoxylanase of Aspergillus steynii IBT 23096 (GenBank accession number: XP_024705365) was up to 88%.

[0034] The codon-optimized nucleotide sequence of the xyn10A gene of Pichia preference Pichia according to the present invention is shown in SEQ ID NO.2. The optimized xyn10A sequence was directly synthesized in Beijing Qingke Biotechnology Co., Ltd., connected to the pPICZαA vector, and transformed into Escherichia coil (E.coil) TOP10 competent cells (Beijing Tiangen Bioche...

Embodiment 3

[0035] Example 3 Construction of High Efficiency Secretion and Expression of Xylanase Pichia pastoris Engineering Strain

[0036] Select a single colony of transformed Escherichia coli and inoculate it in LB liquid medium, culture it on a shaker at 37°C overnight, use a plasmid extraction kit (Omega Company, the United States) to extract a high-purity recombinant cloning plasmid, and use Sac I (TaKaRa Company, Inc., Japan) linearization.

[0037]At the same time, Pichia pastoris X-33 competent cells were prepared. Mix the linearized expression granulation with Pichia pastoris X-33 competent cells, transfer to a 0.2cm ice-cooled electroporation cuvette and click, after clicking, immediately add autoclaved and ice-cooled 1mol Sorbitol solution such as / L, mixed well, cultured at 28°C for 2-3h, coated the bacterial cell suspension on the YPDS solid plate medium containing Zeocin (100μg / mL), and cultivated at 28°C until growth Clear colonies. A single colony was picked and stre...

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Abstract

The invention provides a high-temperature-resistant xylanase gene and application thereof. The gene is from aspergillus sulphureus, and is named as xyn10A, and the sequence of the gene is shown as SEQID NO. 1; the nucleotide sequence of the gene after optimization of a codon preferential to pichia pastoris is shown as SEQ ID NO. 2; based on the xyn10A gene after codon optimization, pichia pastoris engineering bacteria for efficiently secreting and expressing xylanase xyn10A are constructed; the xylanase secreted and expressed by the bacteria has thermal stability, the optimum catalytic temperature is 70 DEG C, the optimum pH value is 5.0, and high relative activity is kept between pH 4.0-8.0; and the enzyme activity of the xylanase xyn10A can be improved by adding metal ions, and the degradation rate of the enzyme acting on xylan for 30min is 20.07%. The xylanase has good application prospects in the paper industry and the feed industry.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a high temperature resistant xylanase gene and its application. Background technique [0002] The components of plant cell walls include cellulose, hemicellulose, and lignin, among which xylan, the main component of hemicellulose, is a rich biomass resource and the most abundant polysaccharide in nature except cellulose. At present, the methods of degrading xylan include physical method, chemical method and biodegradation method. Among them, the physical and chemical methods have their own advantages and disadvantages, but compared with the physical and chemical methods, the enzymatic hydrolysis method has mild reaction conditions and specific reaction. Therefore, the combination of physical method (heat treatment) and chemical method (acid-base treatment) with enzymatic hydrolysis is considered to be the most effective method for degrading xylan. Xylan is a poly-fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N1/19A23K20/189D21C9/10C12R1/84
CPCC12N9/2482A23K20/189D21C9/1036C12Y302/01008
Inventor 曹云鹤刘亚京王剑杨勇智鲍成玲
Owner CHINA AGRI UNIV