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Xylanase gene derived from streptomyces chartreusis and xylanase thereof

A technology of xylanase, SEQ-2, which is applied in the directions of genetic engineering, plant genetic improvement, and microorganism-based methods, and can solve problems restricting the application of xylanase

Active Publication Date: 2016-06-08
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Secondly, the biological activity of xylanase is established in a suitable reaction system, because most of the xylanases can obtain the best catalytic results under relatively mild conditions, and in the paper industry, food processing, textile industry, etc. Extreme conditions such as high temperature and high pressure are often used in industrial production such as industry, feed industry, and bioenergy, which also restricts the further application of xylanase

Method used

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  • Xylanase gene derived from streptomyces chartreusis and xylanase thereof
  • Xylanase gene derived from streptomyces chartreusis and xylanase thereof
  • Xylanase gene derived from streptomyces chartreusis and xylanase thereof

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Embodiment 1

[0034] Example 1: Gene Cloning of the Xylanase XynA of Streptomyceschartreusis L1105

[0035] 1.1 Genomic DNA extraction

[0036] Inoculate Streptomyces elixirs L1105 in LB liquid medium, culture at 30°C, 150rpm for 72h, draw 1.5mL of the bacteria solution, put it in a 2mL centrifuge tube and centrifuge at 10000×g for 5min at room temperature to obtain the bacteria. Bacterial genome was extracted to obtain total xylanase genome DNA ( figure 1 ).

[0037] 1.2 Design of degenerate primers

[0038] In the NCBI protein database (http: / / www.ncbi.nlm.nih.gov / ), enter the 15 amino acid sequences of the N-terminal of xylanase ASTLGAAAAEKGRYF (Ala-Ser-Thr-Leu-Gly-Ala-Ala-Ala- Ala-Glu-Lys-Gly-Arg-Tyr-Phe) was compared by Blastn, and 10 xylanases encoded by Streptomyces with high homology to the xylanase gene of Streptomyces elixirs L1105 were obtained Design primers in the high homology region of the gene sequence. The conserved sequence region ( figure 2 ).

[0039] 1.3PCR ampl...

Embodiment 2

[0059] Embodiment 2: Analysis of recombinant xylanase XynA protein

[0060] 2.1 Construction of pET28a(+) expression vector:

[0061] 2.1.1 PCR amplification gene xynA

[0062] Primers were designed according to xynA, considering the multiple cloning site (MCS) of the expression vector and the sequence of xynA, and restriction sites EcoRI and XhoI were introduced at both ends of xynA. Refer to Table 2.1 for the PCR reaction system. The PCR cycling conditions were: 94°C, 4min; 94°C, 30s; 68°C, 30s; 72°C, 90s; a total of 30 cycles, with a final extension of 10min at 72°C. PCR products were recovered by gel cutting. The primers were synthesized by Beijing Aoke Dingsheng Company, and the primers are listed in Table 3.

[0063] Table 3 Primers for cloning xynA

[0064] Table3PrimersforxynA

[0065]

[0066] 2.1.2 Construction of recombinant plasmid pET28a-xyn4

[0067] The expression vector pET28a and xynA with restriction sites were digested with EcoRI and XhoI, respecti...

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Abstract

The invention discloses a xylanase (Xyn A) gene derived from streptomyces chartreusis. The gene comprises nucleic acid and protein / polypeptide sequence. The xylanase encoded by the gene has 1393 base pairs; the content of G and C is 68%, the gene encodes 460 amino acids, the molecular weight is about 48.2 kDa, the isoelectric point (pI) is 9.04, and the gene contains a section of signal peptide sequence. The xylanase has a typical structure of F / 10 glucoside hydrolase, can translate a section of cellulose binding domain (CBD) with a molecular weight of 10 kDa. The enzyme activity, which is measured by a DNS method, of xylanase is 19.7 + / - 1.2 U / mL. The alkaline xylanase and products thereof can be applied to papermaking industry, textile industry, food processing, biological energy, feed industry, cosmetics, medicine, health products, wine industry, crop production, and various biological reactors.

Description

technical field [0001] The invention relates to a xylanase gene derived from Streptomyceschartreusis L1105 and the application of the product xylanase. Background technique [0002] Xylan is a heterogeneous polysaccharide (heteropolysaeeharides), formed by β-D-xylopyranose units connected by β-1,4-glycosidic bonds. The most abundant plant hemicellulose is xylan, which accounts for 15-35% of plant cells by dry weight. [0003] Xylanase is a kind of glycoside hydrolase, including β-1,4-endoxylanase (EC3.2.1.8), β-xylosidase (EC3.2.1.37), α-L- Furanotype arabinosidase (EC3.2.1.55), α-glucuronidase (Ee3.2.2.1), acetylxylan lipase (EC3.2.1.6), phenolic acid lipase. At present, the most studied xylanase is β-1,4-endoxylanase, which hydrolyzes the main chain of macromolecules to generate xylooligosaccharides and xylose, and is the main force among xylan degrading enzymes. [0004] At present, the main source of xylanase is produced by microorganisms, and the same microorganism c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12N15/63C12N1/21C12N1/19C12N1/15C12R1/52
Inventor 李秀婷熊科朱运平滕超闫子祥
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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