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Eosinophil beta-mannanase MAN5A and gene and application thereof

A technology of mannanase and gene, 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 mannanase, only a few acidic mannanase genes have been cloned, and the use of genetic engineering methods to industrialize the production of acidophilic mannanase products has not been reported.

Method used

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  • Eosinophil beta-mannanase MAN5A and gene and application thereof
  • Eosinophil beta-mannanase MAN5A and gene and application thereof
  • Eosinophil beta-mannanase MAN5A and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1 Screening of acidophilic fungus Bispora sp.MEY-1

[0132] 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, konjac flour 0.5%, pH2.5), spread on the plate of enzyme-producing medium (same enrichment medium, plus 1.5% agarose, pH2.5) after dilution according to routine, and cultivate at 30°C On the 5th to 6th day, the colony that produced the hyaline circle was picked and streaked on the enzyme-producing medium plate, and the streaking and isolation process was repeated for 3 rounds to purify the strain. The strain secreting mannanase was screened by this method.

[0133] 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 and not easy to stir up. Conidiophores erect, (6...

Embodiment 2

[0135] Example 2 Cloning of the gene man5A encoding the acidophilic fungus Bispora sp.MEY-1β-mannanase

[0136] Extraction of acidophilic fungus Bispora sp.MEY-1 genomic DNA:

[0137] 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.

[0138] The degenerate primers P1 and P2 were designed a...

Embodiment 3

[0144] RT-PCR analysis of embodiment 3 β-mannanase gene

[0145] Extract the total RNA of Bispora sp.MEY-1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (MAN5A F: 5′-ATGCTTTTTCAAGTGGGAACAGTGCTTTTATTGGCC-3′, MAN5A R: 5′-TTACACAGGCTTCTCCAGCATTGCCGC-3′) The single-stranded cDNA was amplified to obtain the cDNA sequence of mannanase, and the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0146] After comparing the genome sequence and cDNA sequence of mannanase enzyme, it was found that the gene has 3 introns, 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 man5A was homologously compared with the mannanase gene sequence on GeneBank, the highest amino acid sequence identity was 47.1%, and the nucleotide sequence identity was 45...

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Abstract

The invention relates to the genetic engineering field, especially to a strain Bispora sp.MEY-1 which produces an acidophilic beta-mannanase and the acidophilic beta-mannanase MAN5A got from the strain having an amino acid sequence shown as SEQ ID NO.1 or 2, a gene man5A for coding said beta-mannanase having a nucleotide sequence shown as SEQ ID NO.1 or 2, and a recombinant vector containing said gene and applications. The beta-mannanase of the invention has the following advantages: the optimum pH 1.0-1.5, the optimum temperature 65 DEG C, good pH stability and thermostability, high specific activity, 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 invention relates to the field of genetic engineering, in particular, the invention relates to a bacterial strain Bispora sp.MEY-1 producing acidophilic β-mannanase MAN5A, and the acidophilic β-mannanase obtained from the strain Its gene man5A, the recombinant vector containing the gene and its application. Background technique [0002] β-mannanase (β-mannanase endo-1, 4-β-D-mannanmannohydr-oase EC3.2.1.78) is a kind of hemicellulase that can hydrolyze mannan, which exists in microorganisms, animals and plants . The quantity of plant hemicellulose is second only to cellulose, and mannan is the main component of plant hemicellulose, which is a linear polysaccharide linked by 1,4-β-D-mannopyranoside bonds. Under the action of β-mannan exonuclease and endomannosidase, mannan can be decomposed into mannose. [0003] β-mannanase ( Millward-Sadler, et al Microbiol. Lett. 141, 183-188). Microorganisms are an important source of producing β-mannanase,...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/42C12N15/56C12N15/63C12N15/81C12N1/15C12N1/19C12N1/21A23K1/165A23L1/30A61K38/47C12R1/645A23K20/189A23L29/00
Inventor 姚斌罗会颖王亚茹杨培龙柏映国石鹏君孟昆袁铁铮史秀云
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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