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Alpha-L-fucosidase OUCJdch-16 and application thereof

A technology of oucjdch-16 and fucosidase, which is applied in the field of functional enzymes and can solve the problems that it is difficult to meet industrial production and application, and the enzyme production level of wild-type strains is low.

Active Publication Date: 2021-06-18
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fucosidase can use the substrate pNp-fucose and lactose to synthesize difucosyllactose, but the enzyme production level of the wild-type strain is low, which is difficult to meet the requirements of industrial production and application. Heterologous expression can be used to increase the enzyme production level , to prepare enzyme preparations for industrial production

Method used

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  • Alpha-L-fucosidase OUCJdch-16 and application thereof
  • Alpha-L-fucosidase OUCJdch-16 and application thereof
  • Alpha-L-fucosidase OUCJdch-16 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 α-L-fucosidase gene OUCJDCH-16 cloning

[0035] The α-L-fucosidase OUCJDCH-16 gene of the present invention is obtained by genomic amplification. The inventors excavate to the alpha-L-fucoside fragment of strain Flavobacterium AlgoLA, which is purchased in the China General Microbiological Culture Collection Center, CGMCC, and inventors extracted the genome of the bacteria. OUCJDCH-16 sequences were obtained by gene amplification, which contained 1371 base sequences, such as SEQ ID NO.2, encoded 456 amino acid sequences, as shown in SEQ ID NO.1. According to the evolution tree compared, the alpha-L-fucosidase is found to be the 29th family of glycoside hydrolase (GH29).

[0036] The extracted genome is a template, and the PCR amplification OUCJDCH-16 gene fragment is designed to the upper and downstream of the α-L-fucosidine OUCJDCH-16 gene.

[0037] The sequence of the primer is as follows:

[0038] Upstream primers: 5'-gatccgaattcgagctcccgtatgcaaaamaaatcavaaaaa-...

Embodiment 2

[0043] Example 2 expression vector of 2α-L-fucosidase gene

[0044] The gene fragment is connected to the PET-28A cloning vector to connect to the seamless cloning technique, transfer the ligation product into E.Coli DH5α sensitive cells, coated with (lb) medium solid plate containing 50 μg / ml sulfate. . After 12 to 16 hours of 37 ° C, it was picked up a monoclonal to a 37 ° C oscillating bed containing 50 μg / ml sulfate ltin Liquid medium, and the rotational speed was 220 rpm cent culture overnight, and the positive verification was sequenced, and was named PET28A- OUCJDCH-16.

Embodiment 3

[0045] Example 3 Recombinant plasmid and engineering of engineering bacteria

[0046] Extract the correct recombinant plasmid and transformed into the host E.Coli BL21 sensitive cells, and construct a good engineering bacterium grows on the sulfate sulfate resistance plate.

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Abstract

The invention discloses alpha-L-fucosidase OUCJdch-16 with an amino acid sequence as shown in SEQ ID NO. 1, a gene for coding the alpha-L-fucosidase OUCJdch-16 and with a nucleotide sequence as shown in SEQ ID NO. 2, and application of the alpha-L-fucosidase OUCJdch-16 in the hydrolysis of pNp-Fuc / the preparation of the difucosyllactose. The invention further discloses a recombinant expression vector and a recombinant host containing the gene for coding the alpha-L-fucosidase OUCJdch-16. The alpha-L-fucosidase OUCJdch-16 has certain psychrophilicity, and has relatively high enzyme activity at the temperature of 25-45 DEG C, so that the alpha-L-fucosidase OUCJdch-16 can be industrially applied in a relatively wide temperature range. In addition, a liquid phase result shows that the OUCJdch-16 has a higher conversion rate under the optimal reaction condition, and has good biological catalytic synthesis efficiency.

Description

Technical field [0001] The present invention relates to α-L-fucosidase OUCJDCH-16 and its application thereof, belonging to function enzyme technology. Background technique [0002] Diktrilosylmodose is a human milk hypoglycemia consisting of an alpha (1 → 2) glycosidic linkage, which has a human milk hypoglycemia for regulating intestinal flora and immune system, and resists pathogens. Adhesion, promoting nervous system development and repair and raise physiological activity such as human survival of HIV infections, which makes two fucosamosyl in the fields of medicine, health products, food, agriculture, and therefore has a greater application value, so a lot Synthetic diktrinosamoside is of great significance. [0003] At present, the preparation method of diktrinyl lactose is mainly natural extraction, chemical method, and biological enzyme method. Baby mainly obtains diktellactose from breast milk, but because the content in breast milk is not only related to mother blood ty...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N1/21C12P19/14C12P19/00C12R1/19
CPCC12N9/2402C12N15/70C12P19/14C12P19/00C12Y302/01051
Inventor 毛相朝姜宏周文婷孙建安薛长湖
Owner OCEAN UNIV OF CHINA
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