Laminarinase ouc-l1 and its coding gene and application

A technology of OUC-L1 and laminarinase, applied in laminarinase OUC-L1 and its encoding gene and application field, can solve the problems of biological activity destruction of laminarin, difficult to control the degree of degradation, difficult to separate and purify, etc., and achieve good biological performance. The effect of catalytic efficiency, good stability and high purity

Active Publication Date: 2022-03-22
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 its coding gene and application
  • Laminarinase ouc-l1 and its coding gene and application
  • Laminarinase ouc-l1 and its coding gene and application

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Effect test

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 present invention discloses laminarinase OUC‑L1, the amino acid sequence of which is shown in SEQ ID NO:1, and the nucleotide sequence of the gene encoding the laminarinase OUC‑L1 is shown in SEQ ID NO.2. The laminarinase OUC-L1 of the present invention is excavated from the genome of human intestinal bacteria A.muciniphila, belongs to the glycoside hydrolase GH16 family, can effectively hydrolyze laminarin, and its main product is three-part oligosaccharide, which has a high Specific enzyme activity, with good biocatalytic efficiency. It has the highest reactivity at 35°C. In addition, the enzyme also has good cold adaptability. The invention also discloses an enzyme preparation containing laminarinase, which still has considerable catalytic activity at low temperature and has good potential for industrial application.

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 Patents(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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