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Application of protein AxMan113A as beta-mannase

A technology of mannanase and mannan, applied in the field of microorganisms

Active Publication Date: 2018-06-29
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No other β-mannanases belonging to the glycoside hydrolase 113 family of microbial origin have been reported

Method used

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  • Application of protein AxMan113A as beta-mannase
  • Application of protein AxMan113A as beta-mannase
  • Application of protein AxMan113A as beta-mannase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, the preparation of recombinant β-mannanase

[0074] 1. Construction of recombinant plasmid pET28a-AxMan113A

[0075] 1. Extract the genomic DNA of xylan facultative bacillus NBRC15112 and use it as a template, and use the artificially synthesized upstream primer: 5'-CCG GAATTC ATGGAATTTATTAAGGGTTTTACAT-3' (the underline is the cutting site of the restriction endonuclease EcoRI) and downstream primer: 5'-ATAAGAAT GCGGCCGC TTAACGTGATTGGTAATAAGCTTTA-3' (the underline is the restriction endonuclease NotI restriction endonuclease cutting site) is primer, carries out PCR amplification, obtains PCR amplification product.

[0076] The reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min.

[0077] 2. Take the PCR amplification product obtained in step 1, perform agarose gel electrophoresis, and then recover a DNA fragment of about...

Embodiment 2

[0095] Embodiment 2, the determination of the enzymatic property of recombinant β-mannanase

[0096] Buffer to be tested: pH3.0 citrate-phosphate buffer, pH3.5 citrate-phosphate buffer, pH4.0 citrate-phosphate buffer, pH4.5 citrate-phosphate buffer, pH5.0 lemon Acid phosphate buffer, pH5.5 citrate phosphate buffer, pH6.0 citrate phosphate buffer, pH6.5 citrate phosphate buffer, pH7.0 citrate phosphate buffer, pH3.0 lemon Acid buffer, pH3.5 citrate buffer, pH4.0 citrate buffer, pH4.5 citrate buffer, pH5.0 citrate buffer, pH5.5 citrate buffer, pH6 .0 citrate buffer, pH6.0 phosphate buffer, pH6.5 phosphate buffer, pH7.0 phosphate buffer, pH7.5 phosphate buffer, pH8.0 phosphate buffer, pH8. 0 CHES buffer, pH 8.5 CHES buffer, pH 9.0 CHES buffer, pH 9.5 CHES buffer, pH 10.0 CHES buffer, pH 9.0 glycine-sodium hydroxide buffer, Glycine-NaOH buffer at pH 9.5, Glycine-NaOH buffer at pH 10, Glycine-NaOH buffer at pH 10.5, CAPS buffer at pH 10.0, CAPS buffer at pH 10.5 or CAPS buffer a...

Embodiment 3

[0115] Example 3. Recombinant β-mannanase hydrolyzes locust bean gum to produce mannan oligosaccharides

[0116] Thin-layer chromatography analysis: Spot 2 μL of the sample to be tested on the TLC analysis plate, add the developing agent dropwise for two times, then completely wet it with the color developing solution and dry it, and develop the color at 100°C. The spreading agent is composed of 2 parts by volume of n-butanol, 1 part by volume of ethanol and 1 part by volume of water. The developer consists of 95 parts by volume of methanol and 5 parts by volume of sulfuric acid. The mixture of standard products of mannose, mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose was used as the standard control.

[0117] Reaction system (total volume: 1 mL): pH 6.5, 50 mM citrate phosphate buffer, locust bean gum and the recombinant β-mannanase solution prepared in Example 1 were mixed to obtain a reaction system. In the reaction system, the initial concentrat...

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Abstract

The invention discloses application of protein AxMan113A as beta-mannase. The protein AxMan113A provided by the invention is protein with an amino acid sequence shown as a sequence 2 in a sequence table or protein with an amino acid sequence shown as a sequence 4 in the sequence table. Proved by experiments, the protein AxMan113A has the activity of the beta-mannase, so that vegetable gum, such aslocust bean gum and konjac powder, with rich mannan can be hydrolyzed in a high-efficiency manner; meanwhile, the protein AxMan113A has good acid resistance, heat resistance and hydrolysis characteristic, and is obviously different from the existing beta-mannase. The protein AxMan113A and an encoding gene of the protein AxMan113A have important economic value and practical significance in the industries of foods, feed and the like.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to the application of protein AxMan113A as β-mannanase. Background technique [0002] Mannan is a linear polysaccharide composed of mannose linked by β-1,4-glycosidic bonds, and its main chain also contains glucose residues linked by β-1,4-glycosidic bonds and α-1,6-glycosidic bonds. linked galactose residues. Mannan exists widely in nature and is the most abundant hemicellulose besides xylan (Srivastava and Kapoor. Biotechnology Advances, 2017, 35: 1-19). Mannan is the second largest component of hemicellulose and widely exists in nature. It is the main component of the cell wall of various plants and the main energy storage substance in the endosperm of various plant seeds. Because of its complex structure, the complete degradation of mannan requires the synergy of multiple enzymes, the most important of which is β-mannanase. β-mannanase has a wide range of sources, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12P19/14C12P19/12C12N15/56
CPCC12N9/2491C12P19/12C12P19/14C12Y302/01025
Inventor 江正强李延啸闫巧娟游鑫杨绍青
Owner CHINA AGRI UNIV
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