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High-temperature acid beta-mannase Man5DW1, and gene and application thereof

A mannanase and acidic technology, applied in the field of genetic engineering, can solve the problems of unsatisfactory application, poor thermal stability, low expression level, etc., and achieve the effect of good resistance to metal ions and good heat resistance

Active Publication Date: 2014-08-27
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

[0006] 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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  • High-temperature acid beta-mannase Man5DW1, and gene and application thereof
  • High-temperature acid beta-mannase Man5DW1, and gene and application thereof
  • High-temperature acid beta-mannase Man5DW1, and gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Cloning of embodiment 1β-mannanase encoding gene man5DW1

[0057] Genomic DNA extraction

[0058] 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 at 12,000 rpm for 10 mi...

Embodiment 2

[0064] Example 2 Obtaining of β-mannanase cDNA

[0065] Total RNA was extracted using Oligo(dT) 20 and reverse transcriptase to obtain a strand of cDNA, then design primers F and R (see Table 1) for amplifying the open reading frame, amplify the single-stranded cDNA, obtain the cDNA sequence of mannanase, and amplify to obtain the product After recovery, they were sent to Ruibo Biotechnology Co., Ltd. for sequencing.

[0066] After comparing the genome sequence and cDNA sequence of mannanase, it is found that the gene contains an intron, the cDNA is 1122bp long, encodes 373 amino acids and a stop codon, and the N-terminal 15 amino acids are its signal peptide sequence , the comparison proves that the gene encoding mannanase isolated and cloned from Alternaria sp. is a new gene.

Embodiment 3

[0067] The construction of embodiment 3 β-mannanase engineering strains

[0068] (1) Construction of expression vector and expression in yeast

[0069] Using the cDNA of the mannanase Man5DW1 sequenced correctly as a template, primers F and R (see Table 1) with EcoR I and Not I restriction sites were designed and synthesized, and the coding region of the mature protein of Man5DW1 was analyzed. Amplify. And utilize EcoR I and Not I to digest the PCR product, connect into the expression vector pPIC9 (Invitrogen, San Diego), the sequence of β-mannanase Man5DW1 mature protein is inserted into the downstream of the signal peptide sequence of the above expression vector, and signal peptide The correct reading frame was formed, and the yeast expression vector pPIC9-man5DW1 was constructed to transform 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 preparati...

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Abstract

The invention relates to the field of gene engineering, and particularly relates to a high-temperature acid beta-mannase Man5DW1, and a gene and application thereof. An amino acid sequence of the high-temperature acid beta-mannase Man5DW1 is shown in SEQ ID NO.1 or SEQ ID NO.2. The invention provides a novel mannose gene, and the encoded mannose has acidity, high temperature, good heat resistance and ability of resisting metal ions, and can be applied to the industries such as feeds, foods, medicines and the like. According to the technical scheme disclosed by the invention, the mannase which is excellent in property and applicable to industrial application can be produced 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 acidic β-mannanase Man5DW1 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 and releases a short β -l,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 protection, and the research ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19C12R1/84C12R1/78C12R1/865
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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