Beta-mannase mRmMan5A and encoding gene and application thereof

A coding gene and coding technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of few reports, and achieve the effect of high optimum temperature, low optimum pH, and great application value.

Active Publication Date: 2016-08-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, some agricultural wastes rich in mannan, such as: coconut meal, coffee grounds, palm meal, etc., are also good substrates for the preparation of mannan oligosacchari

Method used

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  • Beta-mannase mRmMan5A and encoding gene and application thereof
  • Beta-mannase mRmMan5A and encoding gene and application thereof
  • Beta-mannase mRmMan5A and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, the directed evolution of β-mannanase gene

[0049] 1. Random mutation of β-mannanase gene (RmMan5A)

[0050] 1. Error-prone PCR

[0051] Sequence analysis of the wild-type gene of Rhizomucor miehei β-mannanase (as shown in Sequence 2 of the Sequence Listing) revealed that the 5' end of the gene contains a sequence encoding a signal peptide consisting of 19 amino acid residues. Error-prone PCR primers RmMan5AF and RmMan5AR were designed according to the mature protein coding region of Rhizomucor miehei β-mannanase (that is, the signal peptide coding sequence was removed). The primer sequences are as follows:

[0052] RmMan5AF: 5'-CGC GGATCC GCTTCTTCGTTTGTCCAGACAAG-3'; the underlined sequence is the recognition site for BamHI digestion; the underlined sequence is identical to the 58th to 80th sequence of sequence 2 or 4 in the sequence listing;

[0053] RmMan5AR: 5'-CCG CTCGAG TCACTTCTTGGCCATGGCATCAGC-3'; the underlined sequence is the XhoI restricti...

Embodiment 2

[0083] Embodiment 2, preparation of β-mannanase and determination of enzymatic properties thereof

[0084] 1. Preparation of recombinant protein

[0085] 1. Induced expression of recombinant protein

[0086] Inoculate recombinant bacteria A or control bacteria into liquid LB medium (containing 50 μg / mL kanamycin) and shake culture (37°C, 200rpm), until OD 600 When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 1 mmol / L, induce culture at 30°C overnight, collect the bacteria at 10,000 × g, resuspend, ultrasonically break, centrifuge at 10,000 × g for 10 min, and collect the supernatant as the crude enzyme solution .

[0087] 2. Purification of recombinant protein

[0088] Recombinant proteins were purified using Sepharose Ni-IDA affinity columns. Specific steps are as follows:

[0089] Use buffer A to equilibrate the Ni-IDA affinity column for 5-10 column volumes, load the crude enzyme solution of recombinant bacteria A or control bacteria obtained...

Embodiment 3

[0118] Example 3, Pichia pastoris high-density fermentation expression of recombinant protein mRmMan5A

[0119] 1. Acquisition of recombinant bacteria

[0120]1. Using the extracted plasmid (containing the mRmMan5A gene) obtained in Example 1 as a template, PCR amplification was performed using mRmMan5AF and mRmMan5AR primer pairs to obtain a PCR amplification product. The primer sequences are as follows:

[0121] mRmMan5AF: 5'-CCATG TACGTA GCTTCTTCGTTTGTCCAGACAAG-3'; the underlined sequence is the SnaBI restriction recognition site; the underlined sequence is identical to the 58th to 80th sequence of sequence 2 or 4 in the sequence listing;

[0122] mRmMan5AR: 5'-CCG CCTAGG TCACTTCTTGGCCATGGCATC-3'; the underlined sequence is the AvrII restriction recognition site; the underlined sequence matches the 1117th to 1137th sequence of sequence 2 or 4 in the sequence listing.

[0123] 2. Carry out double enzyme digestion to the PCR amplified product obtained in step 1 with res...

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Abstract

The invention relates to beta-mannase mRmMan5A and an encoding gene and application thereof .Compared with original beta-mannase mRmMan5A, the beta-mannase mRmMan5A has the advantages that the optimal pH is low and is 4.5, and the optimal temperature is high and is 65 DEG C .Compared with beta-mannase reported in the past, the beta-mannase mRmMan5A has higher application value in the industries of food, feed and the like .The enzyme activity of beta-mannase of engineering bacteria fermentation liquor prepared by introducing the encoding gene of the protein into pichia pastoris can reach 72626 U/mL (protein content is 9.1 mg/mL), and efficient expression is achieved .The beta-mannase is used for hydrolyzing palm meal subjected to steam explosion, hydrolysate mainly contains mannan oligosaccharide with polymerization degree of 2 to 4, the mannan oligosaccharide yield is 19.6%, and the mannan oligosaccharide conversion rate is about 60%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a beta-mannanase mRmMan5 and its coding gene and application. Background technique [0002] β-mannan is a polymer polysaccharide formed by linking mannose through β-1,4-glycosidic bonds, and is the second largest component of hemicellulose. The main chain of β-mannan is usually composed of mannose, and some glucose residues, such as glucomannan, are inserted during the period; in addition, there are also α-1,6-glycosidic bonds in some mannans. Galactose side chains, such as galactomannan and galactoglucomannan (Malgase et al. World Journal of Microbiology and Biotechnology, 2015, 31: 1167-1175). Due to the complex structure of mannan, its complete degradation requires the synergy of various enzymes, such as β-mannanase (EC3.2.1.78), β-mannosidase (EC3.2.1.25), β-glucoside Enzymes (EC3.2.1.21), α-galactosidase (EC3.2.1.23) and mannan acetylesterase (EC3.1.1.6), etc. (Moreira et al. ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/11C12P19/14
Inventor 江正强李延啸闫巧娟徐梦宇易萍刘学强
Owner CHINA AGRI UNIV
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