Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene

A technology of mannanase and ppic9-ba-man5a, which is applied in the field of genetic engineering and can solve the problems of low yield of mannanase and inability to meet industrialized production and the like

Active Publication Date: 2012-02-29
SHANDONG LONGKETE ENZYME PREPARATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the yield of mannanase produced by natural strains

Method used

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  • Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene
  • Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene
  • Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1 Enzyme-producing properties of the fungus Bispora antennata CBS 126.38

[0127] After Bispora antennataCBS 126.38 was cultured in malt extract medium, it was inoculated in induction medium (0.2% MgSO 4 ·7H 2 O, 0.1% KH 2 PO 4 , 0.1% CuSO 4 ·5H 2 O, 0.1% CaCl 2 , 0.5% peptone, 1% corn cob flour, 1% bran, 0.5% konjac flour, pH 5.0), cultivated at 30°C for 3-4 days, and measured its cellulose and hemicellulase activity. It was determined that the bacteria was a high-production mannanase strain.

Embodiment 2

[0128] Example 2 Cloning of the gene ba-man5A encoding the fungus Bispora antennata CBS 126.38β-mannanase

[0129] 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.

[0130] The degenerate primers P1 and P2 were designed and synthesized according to the published conserved sequence of mannan...

Embodiment 3

[0135] RT-PCR analysis of embodiment 3 β-mannanase gene

[0136] Extract the total RNA of Bispora antennata CBS 126.38, use reverse transcriptase to obtain a strand of cDNA, then design appropriate primers M5AF and M5AR (Table 1) to amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify After the product was recovered, it was sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0137] After comparing the genome sequence and cDNA sequence of mannanase enzyme, it was found that the gene has 5 introns, the cDNA is 1299bp long (SEQ ID NO.4), encodes 432 amino acids (SEQ ID NO.1) and a stop codon The 18 amino acids at the N-terminal are its predicted signal peptide sequence, and the nucleotide sequence of the mature protein part of the measured gene ba-man5A is homologously compared with the mannanase gene sequence on GeneBank, which is derived from The putative protein of Botryotinia fuckeliana has the highest sequence identity of 60%, and the hig...

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Abstract

The invention relates to the field of gene engineering, in particular to beta-mannaseBA-Man5A with a wide pH range, a gene thereof and application of the gene. The invention provides the beta-mannaseBA-Man5A with the wide pH range. The beta-mannaseBA-Man5A with the wide pH range has the amino acid sequence shown as SEQ ID NO.1 or 2. The invention also provides a gene for coding the beta-mannaseBA-Man5A with the wide pH range, a recombinant vector and a recombinant strain which comprise the gene, and application of the gene. The nucleotide sequence of the gene is shown as SEQ ID NO. 3 or 4. The beta-mannaseBA-Man5A has high activity in acidic, neutral and alkaline ranges, a wide pH range, high acid and alkali resistance, high heat resistance and excellent anti-protease ability, and can be applied to industries such as animal and fish feed, food, medicines, paper making and the like. According to the technical scheme of the invention, mannase can be produced by a gene engineering method.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a β-mannanase BA-Man5A with a wide pH range and its gene and application. Background technique [0002] Mannan is the main component of plant hemicellulose, a linear polysaccharide linked by 1,4-β-D-mannopyranoside bonds. β-mannanase (β-1,4-D-mannan mannohydrolase, EC3.2.1.78) is a hydrolase that can degrade the main chain of mannan, belonging to the hemicellulase class. β-mannanase has a wide range of sources, and β-mannanase exists in many microorganisms, plants and some lower animals (Millward-Sadler, et al FEMS Microbiol. Lett. 1996.141: 183-188). Microbes are an important source of β-mannanase, which has obvious advantages such as high activity, low cost, stable source, and convenient extraction. However, the yield of mannanase produced by natural strains is low, which cannot meet the needs of industrial production. The use of microbial reactors t...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21C12R1/84
Inventor 郭庆文王兴吉王忠连李芳芳刘文龙孙硕钱娟娟
Owner SHANDONG LONGKETE ENZYME PREPARATION
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