Preparation method of alpha-L-fucosidase

A fucosidase and amino acid technology, applied in the fields of biochemistry, molecular biology, and genetic engineering, can solve the problems of unstable source, poor repeatability, and high price, and achieve high repeatability, high stability, and high yield. Effect

Pending Publication Date: 2020-12-08
海丰生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most AFU products on the market are extracted from animal organs, which have the disadvantages of unstable source, poor repeatability, very high cost and high price

Method used

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  • Preparation method of alpha-L-fucosidase
  • Preparation method of alpha-L-fucosidase
  • Preparation method of alpha-L-fucosidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, design and construction of recombinant vector

[0037] The species source of the α-L-fucosidase in this example is: Fibrella aestuarina BUZ 2, and the Uniprot number is: IOK5U2. The amino acid sequence of the α-L-fucosidase in this embodiment is shown in SEQ ID No.1, and the nucleotide sequence of the gene encoding the α-L-fucosidase is shown in SEQ ID No.2.

[0038] In this example, the part of the amino-terminal signal peptide sequence was removed by artificially synthesizing the full-length gene sequence, and it was connected into the vector pET26b. The pET series vectors (formerly Novagen, now Merck Millipore) used in the present invention have enzyme cleavage sites of NdeI and XhoI . The gene synthesis and vector construction were completed in Sangon Bioengineering (Shanghai) Co., Ltd., and the constructed recombinant vector was named pSF14.

[0039] Transform the plasmid vector pSF14 into Escherichia coli BL21 (DE3) competent cells by chemical meth...

Embodiment 2

[0041] Embodiment 2, expression of α-L-fucosidase

[0042] Pick the strain expressing α-L-fucosidase obtained from the positive transformant, inoculate the recombinant expression strain in 5 ml liquid LB medium, and culture with shaking at 37° C. and 250 rpm for 7-8 hours.

[0043] Transfer the strain culture solution to 100ml LB liquid medium, shake and cultivate overnight at 37°C and 250rpm; the next day, transfer the strain culture solution to two 3L culture flasks, each containing 1L LB medium, 37 Cultivate with shaking at 250 rpm until the absorbance at 600 nm is 0.8-1.0. The inducer IPTG was added to a final concentration of 0.1 mM, and the culture was shaken at 16° C. and 180 rpm for 24 hours. The bacterial cells were collected by centrifugation at 8000 rpm for 10 minutes. The bacterial pellet was resuspended with 20mM Tris-HCl pH8.0 and 200mM NaCl, and ultrasonically disrupted for 40min. After the lysate was centrifuged at high speed at 14000 rpm for 30 min, the sup...

Embodiment 3

[0044] The determination of embodiment 3α-L-fucosidase activity

[0045] The α-L-fucosidase prepared in Example 2 is stored in the storage solution of α-L-fucosidase, and the formula of the storage solution is: 20mM Na 2 HPO 4 , 500mM NaCl, 10mM EDTA, pH6.5, take out a total of 10ml of α-L-fucosidase finished product, dilute 100 times, and use the self-developed kit of Haifeng Bio (Beijing) Technology Co., Ltd. and Hitachi 7180 biochemical analyzer to detect the enzyme Activity, the measured value is 72.99U / L, and the enzyme activity concentration of the finished product of α-L-fucosidase is 7.3U / ml.

[0046] Dilute the finished product of α-L-fucosidase 4 times, measure the absorbance at 280 nanometers with a Miou ultra-micro spectrophotometer, the measured value is 2.123, and the calculated protein concentration is 3.19mg / ml, the finished product of α-L-fucosidase The specific activity is 2.29U / mg.

[0047] After 100 times dilution of the finished α-L-fucosidase product, ...

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Abstract

The invention discloses a preparation method of alpha-L-fucosidase. The fucosidase has the characteristics of (a) or (b) or (c) or (d), wherein the amino acid sequence of (a) is shown as SEQ ID NO: 1,and (a) has the activity (EC 3.2.1.51) of alpha-L-fucosidase; the amino acid sequence of (b) has 70 % or more homology with (a); the nucleic acid sequence of (c) is SEQ ID NO: 2 or the encoded aminoacid sequence of (c) is the nucleic acid sequence of (a); and the amino acid sequence, coded by the nucleic acid sequence, of (d) has 70 % or more homology with (a). According to the method, an escherichia coli expression system is adopted; through optimization of expression conditions and purification conditions, the low-cost, high-purity, high-yield, high-stability and high-repeatability activealpha-L-fucosidase is obtained with the shortest production cycle; and the market application prospects are extremely wide.

Description

technical field [0001] The invention relates to the fields of molecular biology, biochemistry and genetic engineering, in particular to a preparation method of α-L-fucosidase. Background technique [0002] Many proteins and lipids in organisms have glycosylation modifications. These modified oligosaccharides not only participate in the regulation of the structural stability of the modified proteins and lipids, but also participate in the regulation of various functions in cells and intercellular interactions. Interaction. Glycoside hydrolases (EC3.2.1) are a class of enzymes that can hydrolyze glycosidic bonds, which can cut and modify these oligosaccharides, thereby changing their regulatory functions. According to different substrate specificities, glycoside hydrolases can be divided into 195 species (EC3.2.1.1-EC3.2.1.195). α-L-fucosidase has been extracted from many microorganisms, plants, molluscs and mammals. According to different functions, it can be divided into ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12Y302/01051C12N15/70
Inventor 刘迎朱成伟侯爽
Owner 海丰生物科技(北京)有限公司
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