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Mannase gene VbMan26A, recombinant plasmid, recombinant strain, mannase and application thereof

A technology of mannase and recombinant plasmid, applied in the field of genetic engineering, can solve the problems such as rare mannase, and achieve the effect of high activity

Inactive Publication Date: 2021-06-29
WUHAN POLYTECHNIC UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to differences in protein sequences, mannosidases from different microorganisms often have different enzymatic properties, for example, different reaction temperatures and pHs, different temperature, pH and salinity tolerances, and different product types ( The products are mannan oligosaccharides of different lengths), etc., but mannosidases with high activity at low temperature and alkaline are relatively rare

Method used

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  • Mannase gene VbMan26A, recombinant plasmid, recombinant strain, mannase and application thereof
  • Mannase gene VbMan26A, recombinant plasmid, recombinant strain, mannase and application thereof
  • Mannase gene VbMan26A, recombinant plasmid, recombinant strain, mannase and application thereof

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Experimental program
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Effect test

Embodiment 1

[0049] The acquisition of embodiment 1 mannosidase gene VbMan26A

[0050] With the protein sequence (UniProtKB number: A2QKT4) of Aspergillus niger (Aspergillus Niger BK01) mannosidase as query sequence, search the GenBank protein sequence resource bank with the Blastp program, find the mannosidase gene involved in the present invention; The preference of the mannosidase gene codon was optimized and designed to obtain the mannosidase gene VbMan26A, the nucleotide sequence of which is shown in SEQ ID NO:1. Mannosidase gene VbMan26A was synthesized in Suzhou Jinweizhi Co., Ltd. The synthetic mannosidase gene VbMan26A was located on the pU57 vector, and the 5' end and 3' end of the synthetic gene had restriction enzyme cutting sites BamH 1 and Xho 1.

Embodiment 2

[0051] The preparation of embodiment 2 recombinant plasmids

[0052] (1) cutting the mannosidase gene VbMan26A obtained in Example 1 from the pU57 vector with restriction endonucleases BamH 1 and Xho 1 (both purchased from Takara Company), and cutting the gel to recover the cut VbMan26A DNA fragment;

[0053] (2) The VbMan26A DNA fragment was connected to the pET-28a vector (a commercial vector for recombinant expression of foreign genes in Escherichia coli, purchased from Novagen) by T4 DNA ligase (purchased from Takara Company). with kanamycin resistance gene);

[0054] (3) pass the connection product through CaCl 2 coli DH5α (a conventional cloning strain in a biological laboratory, purchased from Invitrogen, and stored in the laboratory of Wuhan University of Light Industry) competent cells. The transformed cells were coated on LB agar plates containing 100 μg / mL kanamycin (purchased from Sigma) for positive transformant screening; the preparation of E. coli DH5α compete...

Embodiment 3

[0056] The preparation of embodiment 3 recombinant strains

[0057] (1) The recombinant plasmid pET-28a-VbMan26A that embodiment 2 obtains adopts CaCl 2 coli BL21 (Rosetta) (purchased from Novagen, which is a common gene engineering expression bacterium in the laboratory, which has chloramphenicol resistance and is stored in the laboratory of Wuhan University of Light Industry) competent cells; The preparation and transformation method of state Escherichia coli BL21 (Rosetta) are identical with the method in embodiment 2 step (3);

[0058] (2) Spread the transformed Escherichia coli BL21 (Rosetta) on the LB agar plate containing kanamycin (100 μg / mL) and chloramphenicol (34 μg / mL, purchased from Sigma Company), where the double antibody The colonies grown on the sex plate were recombinant strains.

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Abstract

The invention discloses a mannase gene VbMan26A, a recombinant plasmid, a recombinant strain, mannase and application thereof. The mannase gene VbMan26A is used for encoding the mannase, the nucleotide sequence of the mannase gene VbMan26A is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded mannase is shown as SEQ ID NO: 2. The mannase gene VbMan26A provided by the invention has the advantages that the mannase encoded by the mannase gene VbMan26A has high activity under low-temperature and alkaline conditions, mannan can be effectively hydrolyzed into mannan oligosaccharide with 2-3 residues, and the mannase gene VbMan26A can be applicable to preparation of probiotic mannan oligosaccharide and can also be used for preparing washing products.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a mannase gene VbMan26A, a recombinant plasmid, a recombinant bacterial strain, mannase and applications thereof. Background technique [0002] Hemicellulose is the second most abundant carbohydrate in nature and is found mainly in plant cell walls. Hemicellulose has a variety of forms, mainly xylan, arabinoxylan, glucomannan, galactoglucomannan and so on. Endo-mannase is the most important enzyme (endo-mannase is usually called mannosidase) that degrades mannan. Mannanase randomly cleaves the β-1,4 glycosidic bonds on the mannan backbone and hydrolyzes mannan into mannan oligosaccharides. Mannosidase has great potential in the utilization and industrial application of hemicellulopolysaccharide resources. [0003] Due to differences in protein sequences, mannosidases from different microorganisms often have different enzymatic properties, for example, different rea...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/24C12N1/21C12P19/12C12P19/00C12P19/14C11D3/386C12R1/19
CPCC12N9/2494C12Y302/01078C12P19/12C12P19/00C12P19/14C11D3/38636
Inventor 韩正刚杨江科谢会芳王丹胡玉婷
Owner WUHAN POLYTECHNIC UNIVERSITY
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