Acid xylanase XYNB, genes and application thereof

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

Inactive Publication Date: 2010-06-09
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no reports on the industrial production of acidophilic xylanase products that have properties such as acid resistance, high temperature resistance, and protein stability, and use genetic engineering methods

Method used

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  • Acid xylanase XYNB, genes and application thereof
  • Acid xylanase XYNB, genes and application thereof
  • Acid xylanase XYNB, genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Example 1 Isolation of acidophilic fungus Bispora sp.X-1

[0118] 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 them on the enzyme-producing medium plate, and repeat the streaking and separation process 3 rounds to purify the strain. The strain that secretes xylanase is screened by this method.

[0119] 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 surface ...

Embodiment 2

[0120] Example 2 Cloning of the gene xynB encoding the acidophilic fungus Bispora sp.X-1 xylanase

[0121] Extraction of acidophilic fungus Bispora sp.X-1 genomic DNA:

[0122] 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.

[0123] Degenerate primers P1, P2 were designed and synthesized...

Embodiment 3

[0132] The RT-PCR analysis of embodiment 3 xylanase gene

[0133] Extract the total RNA of Bispora sp.X-1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (XynB F: 5′-ATGCATGCATTCTCATACTTGGCTGTGGCG-3′, XynB R: 5′-CTAATTCGACACAGTCTGATACGCATTTCCACTCC-3′) The single-stranded cDNA was amplified to obtain the cDNA sequence of xylanase, and the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0134] By comparing the genome sequence and cDNA sequence of xylanase, it was found that the gene has an intron, the cDNA is 693bp long, encoding 231 amino acids, and the N-terminal 19 amino acids are its signal peptide sequence. The deduced amino acid sequence has high sequence similarities with xylanases from Hypocrea jecorina, Penicillium funiculosum and Phanerochaetechrysosporium, which are 57.1, 49.8 and 44.4%, respectively. It was proved that the gene encoding xylanase isolated and cloned from Bispora sp...

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Abstract

The invention relates to the field of gene engineering, in particular to acid xylanase XYNB, genes and application thereof. A strain Bispora sp. X-1 of acid producing xylanase is separated, the acid xylanase which has an amino acid sequence expressed as SEQ ID NO.1 or 2 is obtained from the strain, the genes for coding the xylanase is obtained, and the genes have a nucleotide sequence expressed as SEQ ID NO.3 or 4. The xylanase of the invention has the following properties: at the optimal pH of 2.6 and the optimal temperature of 65 DEG C, the xylanase has good pH stability and thermal stability; the specific activity is 2,049 U/mg; and the xylanase has extremely good proteinase resistance and is suitable for industrialized fermentation production. The xylanase serving as a novel enzyme preparation can be widely used in the industries of animal feed, food, medicine, wine brewing, energy and the like.

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.X-1 producing acid xylanase, and the acid xylanase obtained from the bacterial species and its gene, including the gene Recombinant vectors and applications. Background technique [0002] Hemicellulose is 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). Xylan is the main type of hemicellulose (Collins et al..FEMS Microbiol Rev.29:3~23.2005), widely present in agricultural by-products such as corncobs, wheat bran, rice bran, straw, bagasse, etc. However, 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 o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/42C12N15/56C12N15/81C12N1/19C12R1/645
Inventor 史宝军胡爱红罗长财
Owner GUANGDONG VTR BIO TECH
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