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A neutral β-mannanase man26dw1 and its gene and application

A technology of mannanase, ppic9-man26dw1, applied in the field of genetic engineering, can solve the problems of poor heat resistance, low pH value and pH stability of β-mannanase, and achieve good heat resistance and excellent protease resistance Ability, effect over a wide pH range

Active Publication Date: 2016-05-11
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

Compared with bacteria, fungal-derived β-mannanase has a lower optimum reaction pH and pH stability, and its heat resistance is worse than that of bacteria

Method used

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  • A neutral β-mannanase man26dw1 and its gene and application
  • A neutral β-mannanase man26dw1 and its gene and application
  • A neutral β-mannanase man26dw1 and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Cloning of embodiment 1β-mannanase encoding gene man26DW1

[0053] The fungus Alternariasp. was isolated from the soil of Snow Lotus in Tianshan Mountains. Extract fungal genomic DNA:

[0054] Filter the mycelium cultured in liquid for 3 days with sterile filter paper, put it into a mortar, add 2mL extract, grind for 5min, then put the grinding solution in a 50mL centrifuge tube, lyse in a water bath at 65°C for 20min, mix every 10min Homogenize once and centrifuge at 10000rpm for 5min at 4°C. The supernatant was extracted in phenol / chloroform to remove impurities, 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 appropriate amount of TE, and stored at -20°C for later use.

[0055] The degenerate primers P1 and P2 were designed and s...

Embodiment 2

[0059] RT-PCR analysis of embodiment 2β-mannanase gene

[0060] Extract total RNA, use reverse transcriptase to obtain a strand of cDNA, and then design appropriate primers (F: 5'-TGGAATTCCAATCAGTGACCTACCAGGCTG-3, R: 5'-TGGCGGCCGCTCAAGCTGATGTATTCCCATTCTTCCAG-3') to amplify the single-stranded cDNA to obtain mannan The cDNA sequence of the carbohydrase was amplified and recovered, and then sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0061] After comparing the genome sequence and cDNA sequence of mannanase enzyme, it was found that the gene has an intron, the cDNA is 1404bp long, encodes 467 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 man26DW1 was homologously compared with the mannanase gene sequence on GeneBank, and it was confirmed that the gene encoding mannanase isolated and cloned from Alternariasp. was a new gene.

Embodiment 3

[0062] Example 3 Preparation of recombinant β-mannanase.

[0063] The expression vector pPIC9 is subjected to double enzyme digestion (EcoRI+NotI), and the gene man26DW1 encoding mannanase is double enzyme digested (EcoRI+NotI) at the same time, and the gene fragment encoding mature mannanase is cut out and connected to the expression vector pPIC9, The recombinant plasmid pPIC-man26DW1 containing the mannanase gene man26DW1 was obtained and transformed into Pichia GS115 to obtain the recombinant Pichia strain GS115 / man26DW1.

[0064] The recombinant plasmid of mannanase gene man26DW1 containing signal peptide coding sequence was constructed in the same way.

[0065] Take the GS115 strain containing the recombinant plasmid, inoculate it in 400mL of BMGY culture medium, shake it at 250rpm at 30°C for 48 hours, and collect the bacteria by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 72 hours of induction, the supernatant was collec...

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Abstract

The invention relates to the field of genetic engineering and in particular relates to a neutral beta-mannase Man26DW1 as well as a coding gene and application thereof. The neutral beta-mannase Man26DW1 contains an amino acid sequence shown in SEQ ID NO.1 or 2. The beta-mannase Man26DW1 has the properties that optimum pH is 6.0, the beta-mannase Man26DW1 is faintly acid, and enzyme activity is maintained to be more than 40% in the pH range of 4.5-7.5; optimum temperature is 50 DEG C; the enzyme activity remains stable when the beta-mannase Man26DW1 is treated for one hour at the temperature of 50 DEG C; pH stability and thermal stability are good; specific activity is 1,046U / mg, and industrialization fermentation production is easy; the beta-mannase Man26DW1 is used as a novel enzymic preparation and can be widely applied to industries of animal and fish feed, food, medicine, brewing, papermaking and washing agent.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, a neutral β-mannanase Man26DW1 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-mannanmannohydrolase, EC3.2.1.78) is a hydrolase that can degrade the main chain of mannan and belongs 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 high activity, low cost, stable s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84
CPCC12N9/2494C12N15/815C12Y302/01078
Inventor 姚斌罗会颖王彩虹黄火清柏映国石鹏君王亚茹杨培龙孟昆师霞
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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