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Acidic xylanase XYL10A and gene and application thereof

A technology of acid xylanase and xylanase, applied in the field of genetic engineering

Active Publication Date: 2009-06-17
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In my country, there are very few studies on acidophilic xylanase, and only a few acidophilic xylanase genes have been cloned, and the use of genetic engineering methods to industrialize the production of acidophilic xylanase products has not been reported.

Method used

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  • Acidic xylanase XYL10A and gene and application thereof
  • Acidic xylanase XYL10A and gene and application thereof
  • Acidic xylanase XYL10A and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1 Isolation of acidophilic fungus Bispora sp.MEY-1

[0123] After the uranium mine wastewater sample from a mine in Jiangxi was enriched and cultured (enrichment medium: (NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O 0.5g / L, FeSO 4 ·7H 2 O 0.01g / L, CaCl 2 0.2g / L, 0.5% corncob powder, 0.5% bran, pH2.5), and spread it on the enzyme-producing medium ((NH 4 ) 2 SO 4 5g / L, KH 2 PO 4 1g / L, MgSO 4 ·7H 2 O 0.5g / L, FeSO 4 ·7H 2 O 0.01g / L, CaCl 2 0.2g / L, 1% xylan, 1.5% agarose, pH 2.5) on a plate, culture at 30°C for 5-6 days, pick colonies that produce transparent circles, and streak and separate on the enzyme-producing medium plate, repeat streaking and separation Process 3 rounds to purify the strain. The strain that secretes xylanase is screened by this method.

[0124] This strain was cultured on PDA at 30°C for 7 days, and the colony diameter was 2-3cm, gray-black or gray-brown, circular and radial, with velvet-like wrinkles on the sur...

Embodiment 2

[0125] Example 2 Cloning of the gene xyl10A encoding the xylanase of the acidophilic fungus Bispora sp.MEY-1

[0126] Extraction of acidophilic fungus Bispora sp.MEY-1 genomic DNA:

[0127] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL of extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, and mix every 10min. Homogenize once and centrifuge at 10,000 rpm for 5 min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuum, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.

[0128] Degenerate primers P1, P2 were designed an...

Embodiment 3

[0136] RT-PCR analysis of embodiment 3 xylanase gene

[0137] Extract the total RNA of Bispora sp.MEY-1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (Xyl10AF: 5′-ATGTCTTTTCCACTCGCTTCTAATCTCAGGTCTTC-3′, Xyl10AR: 5′-TCATGGACTTTCCGCCTTATGTTGCAAAGCC-3′) to amplify The single-stranded cDNA is obtained from the cDNA sequence of xylanase, and the amplified product is recovered and then sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0138] By comparing the genome sequence and cDNA sequence of xylanase, it was found that the gene has an intron, the cDNA is 1275bp long, encodes 424 amino acids and a stop codon, and the N-terminal 18 amino acids are its predicted signal peptide sequence. The measured nucleotide sequence of the mature protein part of the gene xyl10A was homologously compared with the xylanase gene sequence on GeneBank, and the highest identity was 57.1%, and the highest amino acid sequence identity was 45.7%, which pr...

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Abstract

The invention relates to the genetic engineering field, especially to a strain Bispora sp.MEY-1 which produces an acidic xylanase and the acidic xylanase XYL10A got from the strain having an amino acid sequence shown as SEQ ID NO.1 or 2, a gene for coding said xylanase having a nucleotide sequence shown as SEQ ID NO.1 or 2, and application of a recombinant vector, a recombinant strain and a recombinant enzyme containing said gene. The xylanase of the invention has the following advantages: the optimum pH 2.4, the optimum temperature 80 DEG C, good pH stability and thermostability, specific activity 5437 U / mg, good protease resistance and easy for industrial fermentation production. The product of the invention can be widely used for the animal feeding-stuffs, the food, the medicament, the brewing and the energy industry as a novel enzyme preparation.

Description

technical field [0001] The present invention relates to the field of genetic engineering, specifically, the present invention relates to a bacterial strain Bispora sp.MEY-1 producing acid xylanase, and the acid xylanase XYL10A obtained from the bacterial species and its gene, comprising the Recombinant vectors and applications of genes. Background technique [0002] Xylan is an important component of hemicellulose, the second most abundant polysaccharide in nature after cellulose, accounting for almost one-third of the earth's renewable organic carbon content (Prade.Biotech.and Gentic Engi. Rev.. 13(12): 101-131, 1995). Widely present in agricultural by-products such as corncobs, wheat bran, rice bran, straw, bagasse, etc., but this important renewable resource has been difficult to be effectively utilized. Xylanase is a general term for a class of enzymes that can degrade xylan into oligosaccharides and xylose. The research on xylanase has begun as early as the 1960s, and...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12N15/56C12N15/63C12N15/81C12N1/15C12N1/19C12N1/21A23K1/165A23L1/30C12R1/645A23K20/189A23L33/10
Inventor 姚斌罗会颖杨培龙王亚茹柏映国孟昆袁铁铮石鹏君史秀云
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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