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A kind of β-mannanase mrmman5a and its coding gene and application

A technology for encoding genes and amino acids, applied to β-mannanase mRmMan5 and its encoding genes and application fields, can solve the problems of few reports and other problems, and achieve the effects of high optimum temperature, high-efficiency expression, and great application value.

Active Publication Date: 2019-08-27
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 oligosaccharides, but there are relatively few reports (Chiyanzu et al.Applied Biochemistry and Biotechnology, 2014,172:3538-3557; Ghosh et al. Molecular Biotechnology,2015,57:111-127)

Method used

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  • A kind of β-mannanase mrmman5a and its coding gene and application
  • A kind of β-mannanase mrmman5a and its coding gene and application
  • A kind of β-mannanase mrmman5a and its coding gene and application

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

Provided are a β-mannanase mRmMan5A and an encoding gene and an application thereof The present β-mannanase mRmMan5A has the following advantages compared to original β-mannanase mRmMan5A: optimal pH 4.5 and optimal temperature 65° C. Introducing the present protein encoding gene into pichia pastoris achieves highly effective expression. The present β-mannanase mRmMan5A is used in palm meal having undergone hydrolytic steam explosion processing, the hydrolysate mainly comprising a mannan oligosaccharide having a polymerisation degree of 2-4, the mannan oligosaccharide yield being 19.6%, and the mannan conversion rate being 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 (Malgas 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 (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), β-glucosidase ( EC 3.2.1.21), α-galactosidase (EC 3.2.1.23) and mannan acetylesterase (EC 3.1.1.6), etc. (Moreira et al.Appl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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