A high temperature high specific activity acidic β-mannanase and its gene and application

A technology of mannanase and high ratio, applied in the field of genetic engineering, can solve problems such as poor thermal stability, low expression level, and inappropriate pH range

Active Publication Date: 2016-01-20
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

At present, at home and abroad, although many β-mannanases have been cloned and isolated and their properties are determined, there are some defects in the properties and characteristics of these enzymes, for example, the pH range is not suitable, the thermal stability is poor, and the expression level is low. Meet the needs of practical applications

Method used

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  • A high temperature high specific activity acidic β-mannanase and its gene and application
  • A high temperature high specific activity acidic β-mannanase and its gene and application
  • A high temperature high specific activity acidic β-mannanase and its gene and application

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Experimental program
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Effect test

Embodiment 1

[0134] Cloning of embodiment 1β-mannanase encoding gene man5A

[0135] Extraction of Talaromycesleycettanus Genomic DNA

[0136] The bacteria cultured in liquid for 3 days were centrifuged at 12,000rpm for 10min, and the collected mycelium was added to a high-temperature sterilized mortar, and quickly ground to powder with liquid nitrogen, and then the ground bacteria were transferred to a new, packed Put 15ml of CTAB lysate in a 50mL centrifuge tube, mix it up and down gently, place it in a 70°C water bath for 3 hours, and mix it upside down and gently once every 20 minutes, so as to fully lyse the bacteria. Centrifuge at 12,000 rpm at 4°C for 10 min, pipette the supernatant into a new centrifuge tube, add an equal volume of chloroform for extraction, and place at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Take the supernatant and add an equal volume of phenol / chloroform for extraction, and place it at room temperature for 5 minutes. Centrifuge...

Embodiment 2

[0141] Example 2 Obtaining of β-mannanase cDNA

[0142] Extract total RNA from Talaromycesleycettanus using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, and then design primers F and R to amplify the open reading frame (see Table 1), amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify the product After recovery, they were sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0143]After comparing the genome sequence and cDNA sequence of mannanase, it is found that the gene contains 3 introns, the cDNA is 1296bp long, encodes 431 amino acids and a stop codon, and the N-terminal 18 amino acids are its signal peptide sequence , the comparison proves that the gene encoding mannanase isolated and cloned from Talaromycesleycettanus is a new gene.

Embodiment 3

[0144] The construction of embodiment 3 β-mannanase engineering strains

[0145] (1) Construction of expression vector and expression in yeast

[0146] Using the correctly sequenced mannanase Man5A cDNA as a template, primers F and R with EcoRI and NotI restriction sites were designed and synthesized (see Table 1) to amplify the coding region of the mature protein of Man5A . And use EcoRI and NotI to digest the PCR product, connect it into the expression vector pPIC9 (Invitrogen, SanDiego), and the sequence of the mature protein of β-mannanase Man5A is inserted into the downstream of the signal peptide sequence of the above expression vector to form a correct sequence with the signal peptide. The reading frame was constructed into a yeast expression vector pPIC9-man5A, and transformed into Escherichia coli competent cell JM109. The positive transformants were subjected to DNA sequencing, and the transformants with the correct sequence were used for large-scale preparation of...

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Abstract

The invention relates to the field of genetic engineering, specifically to high-temperature high-specific activity acidic beta-mannanase Man5A and a coding gene and application thereof. The amino acid sequence of the Man5A is represented by SEQ ID No. 1 or SEQ ID No. 2. The invention provides a novel mannanase gene; and mannanase coded by the gene has the advantages of acidity, high temperature, high specific activity, good heat resistance and good antiprotease capacity and can be used in the industries like feeds, food, medicines. With a technical scheme provided by the invention, production of mannanase with excellent properties and industrial applicability can be realized by using genetic engineering means.

Description

technical field [0001] The invention relates to the field of genetic engineering. Specifically, the present invention relates to a high temperature and high specific activity acidic β-mannanase Man5A and its gene and application. Background technique [0002] Plant cell walls are mainly composed of cellulose, hemicellulose, and lignin. Mannan is an important component of plant hemicellulose. It is a linear polymer connected by β-1,4-D-mannose. The side chains of the polysaccharide mainly contain glucosyl, acetyl and hemicellulose. Lactosyl and other substituent groups. β-mannanase (β-mannanase EC3.2.1.78) is an endohydrolase that hydrolyzes mannan. It degrades the β-1,4 glycosidic bond of the mannose backbone in an endo-cutting manner, releasing a short β- 1,4 Mannan oligosaccharides. [0003] In recent years, with the discovery of the physiological functions of mannan oligosaccharides, the rise of green feed, the enhancement of people's awareness of environmental protec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84C12R1/865C12R1/78C12R1/645
CPCC12N9/2494C12Y302/01078
Inventor 姚斌罗会颖王彩虹王亚茹孟昆石鹏君黄火清柏映国杨培龙
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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