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Alkali-resisting beta-mannase Man5A as well as gene and applications thereof

A mannanase and gene technology, applied in the field of genetic engineering, can solve the problems of inability to meet industrial production, low mannanase yield and the like

Active Publication Date: 2013-06-26
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

However, the yield of mannanase produced by natural strains is low, which cannot meet the needs of industrial production

Method used

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  • Alkali-resisting beta-mannase Man5A as well as gene and applications thereof
  • Alkali-resisting beta-mannase Man5A as well as gene and applications thereof
  • Alkali-resisting beta-mannase Man5A as well as gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Cloning of embodiment 1β-mannanase encoding gene man5A

[0105] Fungus Y1 was isolated from forest soil in Hebei. Extract the genomic DNA of the fungus Humicola insolens Y1:

[0106] 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.

[0107] The degenerate primers P1 and P2 were des...

Embodiment 2

[0112] RT-PCR analysis of embodiment 2β-mannanase gene

[0113] Extract the total RNA of Humicola insolens Y1, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (MAN5A F: 5′-ATGCACATCTCGACTGCAAGGTTGCTGACC-3′, MAN5A R: 5′-CTAGTGAGCGTGACGGTTAAGTGCGTTCATC-3′) to amplify the Single-stranded cDNA, the cDNA sequence of mannanase was obtained, the amplified product was recovered and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0114] After comparing the genome sequence and cDNA sequence of mannanase enzyme, it was found that the gene has 2 introns, the cDNA is 1233bp long, encoding 410 amino acids and a stop codon, and the N-terminal 20 amino acids are its predicted signal peptide sequence , the measured partial nucleotide sequence of the mature protein of the gene man5A was homologously compared with the sequence of the mannanase gene on GeneBank. Most of the genes had high sequence identity with the hypothetical protein, but with t...

Embodiment 3

[0115] Example 3 Preparation of recombinant β-mannanase.

[0116] The expression vector pPIC9 is subjected to double enzyme digestion (EcoRI+NotI), and at the same time, the gene mannanase encoding mannanase is double enzyme digested (EcoRI+NotI), and the gene fragment encoding mature mannanase is cut out and connected to the expression vector pPIC9, The recombinant plasmid pPIC-man5A containing Humicola insolens Y1 mannanase gene man5A was obtained and transformed into Pichia pastoris GS115 to obtain recombinant Pichia pastoris strain GS115 / man5A.

[0117] The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 72 hours of induction, the supernatant was collected by centrifugation. The activity of mannanase was determined. The expression level of recombinant mannanase was 15U / mL. SDS...

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Abstract

The invention relates to the field of gene engineering, particularly relating to an alkali-resisting beta-mannase Man5A as well as a gene and applications thereof. The alkali-resisting belt-mannase Man5A comprises an amino acid sequence shown as SEQ ID No.1 or SEQ ID No.2; the gene man5A of the beta-mannase, a recombination carrier containing the gene and applications are coded, wherein the gene man5A comprises a nucleotide sequence shown as SEQ ID No.1 or SEQ ID No.2. The beta-mannase provided by the invention has the following properties: the optimum pH value is 5.5, the beta-mannase has strong alkali resistance; the enzyme activities are respectively 45% and 36% of the highest enzyme activity at the condition that the pH values are 8.0 and 9.0; the optimum temperature is 70 DEG C; the stability and thermostability of the pH value are good; the specific activity is 1,122U.mg<-1>; and the alkali-resisting beta-mannase Man5A has extremely good prolease resistance and is easy for industrial fermentation production; and used as a novel enzymic preparation, the alkali-resisting beta-mannase Man5A can be widely used in animal and fish feeds, food and medicines, and also used in the industries of brewing, paper making and detergents.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an alkali-resistant β-mannanase Man5A 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 been widely used in many fields such as food, feed, medicine, papermaking, textile printing and dyeing, petroleum exploration, fine chemical industry and biotechnology. It is a new type of industrial enzyme with great potential application value. [0003] 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42
Inventor 姚斌罗会颖黄火清王亚茹石鹏君柏映国杨培龙袁铁铮孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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