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Laminarinase OUC-L1 and encoding gene and application thereof

A technology of OUC-L1 and lamina polysaccharase, applied in the field of lamina polysaccharide OUC-L1 and its coding gene and application, can solve the problems of lamina polysaccharide biological activity destruction, difficult to control the degree of degradation, difficult to separate and purify, and achieve good industrialization Application potential, good biocatalytic efficiency, and good stability

Active Publication Date: 2020-06-26
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because chemical and physical methods randomly destroy the glycosidic bonds in laminarin under severe conditions, the degree of degradation is difficult to control, and a series of oligosaccharide products with dispersed polymerization degrees will be formed, which are difficult to separate and purify; moreover, chemical methods and The severe reaction conditions in the preparation of oligosaccharides by physical methods will not only destroy the original biological activity of laminarin, but also often accompany high energy consumption and potential risks of environmental pollution

Method used

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  • Laminarinase OUC-L1 and encoding gene and application thereof
  • Laminarinase OUC-L1 and encoding gene and application thereof
  • Laminarinase OUC-L1 and encoding gene and application thereof

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Embodiment 1

[0024] Example 1 Cloning of Laminarinase OUC-L1

[0025] The enzyme-producing gene of laminarinase OUC-L1 of the present invention is based on human intestinal bacterium A.muciniphila (gifted by Mr. Pang Xiaoyang, Institute of Agricultural Products Processing, Chinese Academy of Agricultural Sciences) genome (although the genome sequence of this bacterium has been disclosed, but about this The laminarinase OUC-L1 gene involved in the invention has not been isolated or amplified separately and its biological activity has been characterized; this invention is the first time that OUC-L1 has been isolated from the genome of human intestinal microorganism Akkermansia muciniphila gene) as a template, obtained through PCR specific amplification:

[0026] Download the human intestinal bacterium A.muciniphila genome sequence (Accession number: GCA_000020225.1) from NCBI, and use the BioEdit software to convert the characterized laminarin enzyme gene sequence such as LamC (Accession num...

Embodiment 2

[0034] Embodiment 2 Containing the expression vector construction of laminarinase gene

[0035] The gene fragment and the pET28a(+) cloning vector were connected by seamless cloning technology, and the connection product was transferred into E.coli DH5α competent cells, and spread on the LB medium solid plate containing 50 μg / mL kanamycin. After culturing in a 37°C incubator for 12-16 hours, pick a single clone into LB liquid medium containing 50 μg / mL kanamycin, culture overnight on a shaker at 37°C at a speed of 220 rpm, and perform sequencing after PCR positive verification. The bacterial liquid verified by sequencing was placed in a glycerol tube for storage at -20°C, and the expression vector was named pET28a-OUC-L1.

Embodiment 3

[0036] Embodiment 3 Containing the recombinant plasmid of laminarinase gene and the construction of engineering bacteria

[0037] The recombinant pET28a-OUC-L1 plasmid was obtained by extracting the bacterial liquid with correct sequencing, which was transformed into the host E.coli BL21 competent cells, and the constructed engineering bacteria were grown on the plate containing 50 μg / mL kanamycin resistance.

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Abstract

The invention discloses laminarinase OUC-L1. The amino acid sequence of the laminarinase OUC-L1 is shown as SEQ ID NO:1, the gene of the laminarinase OUC-L1 is encoded, and the nucleotide sequence ofthe laminarinase OUC-L1 is shown as SEQ ID NO. 2. The laminarinase OUC-L1 is explored from a human intestinal bacterium A. muciniphila genome, belongs to a glycoside hydrolase GH16 family, and can effectively hydrolyze laminarin, and the main product of the laminarinase OUC-L1 is three parts of oligosaccharides, has high specific enzyme activity and good biological catalysis efficiency, and has the highest reaction activity at the temperature of 35 DEG C. In addition, the laminarinase OUC-L1 also has good cold adaptability. The invention further discloses an enzyme preparation containing the laminarinase OUC-L1. The enzyme preparation still has considerable catalytic activity at low temperature and has good industrial application potential.

Description

technical field [0001] The invention relates to laminarinase OUC-L1 and its coding gene and application, and belongs to the technical field of functional gene cloning and expression. Background technique [0002] Laminarin oligosaccharides are the degradation products of laminarin, and the degree of polymerization (DP) is generally between 2 and 10. Studies have confirmed that laminarin oligosaccharides not only have a series of important physiological activities such as improving the body's immunity, regulating intestinal flora, and improving diabetes symptoms, but also have better anti-tumor and antioxidant activities than laminarin. In the current research, enzymatic, physical and chemical methods can be used to degrade laminarin to obtain a series of oligosaccharide products. Chemical method can use acid-base solution to degrade laminarin, while physical method can use γ-ray to degrade laminarin. Because chemical and physical methods randomly destroy the glycosidic bon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/14C12P19/00
CPCC12N9/2402C12P19/14C12P19/00
Inventor 毛相朝姜宏黄倚孙建安薛长湖
Owner OCEAN UNIV OF CHINA
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