High-specific activity acidic beta-mannase Man5D as well as gene and application thereof

A mannanase and acidic technology, applied in the field of genetic engineering, can solve problems such as poor thermal stability, inappropriate pH range, and low expression level, and achieve good heat resistance, high specific activity, and good protease resistance Effect

Active Publication Date: 2014-03-19
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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  • High-specific activity acidic beta-mannase Man5D as well as gene and application thereof
  • High-specific activity acidic beta-mannase Man5D as well as gene and application thereof
  • High-specific activity acidic beta-mannase Man5D as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Cloning of embodiment 1β-mannanase encoding gene man5D

[0053] Extraction of Staphylotrichum coccosporum genomic DNA

[0054] Staphylotrichum coccosporum NBRC31817 was purchased from NBRC (NITE biological resource center) in Japan.

[0055] The bacteria cultured in the enzyme-producing medium for 3 days were centrifuged at 12,000 rpm for 10 minutes, 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 50mL centrifuge tube filled with 15ml CTAB lysate, gently invert up and down to mix, place in a 70°C water bath for 3 hours, every 20min, invert up and down and gently mix once 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...

Embodiment 2

[0060] Example 2 Obtaining of β-mannanase cDNA

[0061] Extraction of Staphylotrichum coccosporum total RNA 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.

[0062] After comparing the genome sequence and cDNA sequence of mannanase, it was found that the gene contained 2 introns, the cDNA was 1116 bp long, encoded 371 amino acids and a stop codon, and the N-terminal 21 amino acids were its signal peptide sequence. The comparison proves that the gene encoding mannanase isolated and cloned from Staphylotrichum coccosporum is a new gene.

Embodiment 3

[0063] The construction of embodiment 3 β-mannanase engineering strains

[0064] (1) Construction of expression vector and expression in yeast

[0065] Using the correctly sequenced mannanase Man5D cDNA as a template, the expression primers F and R with EcoR I and Not I restriction sites were designed and synthesized (see Table 1) for the coding region of the mature protein of Man5D Amplify. And use EcoR I and Not I to cut the PCR product, connect into expression vector pPIC9 (Invitrogen, San Diego), the sequence of β-mannanase Man5D 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-man5D 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 preparation of recombinant plasmids...

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Abstract

The invention relates the field of genetic engineering, and in particular relates to high-specific activity acidic beta-mannase Man5D as well as a gene and an application thereof. The amino acid sequence of the mannase is as shown in SEQ ID NO.1 or SEQ ID NO.2. The mannase provided by the invention has the following properties: the optimal pH value is 5.0, over 60% of the enzyme activity of the mannase can be maintained within the pH value of 3.5-8.0, the optimal reaction temperature is 65 DEG C, the enzyme activity is very stable at 50 DEG C, and in addition, the specific activity is 3133 U mg<-1>. The mannase has acidity, high specific activity and good heat resistance, and can be applied to industries such as feeds, foods and medicines. According to the technical scheme, mannase which is excellent in property and is applicable to industrial application can be produced by using genetic engineering methods.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to a high specific activity acidic β-mannanase Man5D 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 on t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/81C12N1/19
CPCC12N9/2494C12Y302/01078
Inventor 姚斌罗会颖王彩虹石鹏君黄火清王亚茹杨培龙柏映国孟昆
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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