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Core fucosidase as well as preparation and application thereof

A technology of fucosidase and fucose, which is applied in the field of glycobiology to achieve the effect of simple operation and high enzyme cutting efficiency

Pending Publication Date: 2018-09-14
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although the removal of core fucose is of great significance: for example, the removal of coreα1,6 fucose on IgG significantly improves the ADCC effect of antibodies by 50-100 times, while the coreα1,3 fucose on allergens The removal of glycosides has great potential application value for the treatment of allergies, but so far, there have been no reports and applications of glycosidases that can directly and effectively hydrolyze glycoprotein core fucose (including core α1,3 and core α1,6 fucose)

Method used

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  • Core fucosidase as well as preparation and application thereof
  • Core fucosidase as well as preparation and application thereof
  • Core fucosidase as well as preparation and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0067] Preparation of reaction substrate

[0068] Use three types of substrates to carry out the proof of this enzymatic activity among the present invention,

[0069] 1. The chromogenic substrate pNP-α-L-Fuc was purchased from carbosynth, UK;

[0070] 2.3-F, Lewis X oligosaccharides were purchased from carbosynth company in the UK;

[0071] 3. Glycoprotein horseradish peroxidase (HRP) was purchased from sigma;

[0072] The substrate pNP-α-L-Fuc and various oligosaccharides were reconstituted with ultrapure water as a preservation solution, and the glycoprotein HRP was prepared at a concentration of 10mg / ml for preservation;

[0073] The chromogenic substrate pNP-α-L-Fuc and oligosaccharides do not need to be heat-treated, and HRP is prepared with 25mM sodium dihydrogen phosphate solution (pH=4.6) at a concentration of about 0.5mg / ml, and DTT is added to make the final concentration of 10mM, heating at 93°C for 10min, thermal denaturation;

[0074] 2. Enzyme digestion reac...

Embodiment 1

[0088] Example 1: Fucosidase Activity Identification and Enzymatic Properties Experiment

[0089] 1. Identification of fucosidase activity

[0090] Preparation of the reaction substrate: the substrate pNP-α-L-Fuc lyophilized powder (purchased from carbosynth company) was rethawed with distilled water at a concentration of 10 mM, and stored as a preservation solution.

[0091] Enzyme digestion reaction buffer: 25mM sodium dihydrogen phosphate (PH4.5-5.0)

[0092] Enzyme digestion system: Substrate pNP-α-L-Fuc preservation solution 10ul, enzyme 1ul, add phosphate buffer to make up to 60ul volume. In the table below, No. 1 is the experimental group, and No. 2 is the control group.

[0093] Table 1. Configuration of reaction system

[0094]

[0095] Reaction conditions: 37°C, 1h, immediately after the reaction, add 1M Na 2 CO 3 90ul, stop reaction, and carry out OD=405nm measurement with microplate reader;

[0096] Based on the above results, it was judged whether the en...

Embodiment 2

[0111] Example 2: Enzyme digestion experiment of oligosaccharides

[0112] Substrate configuration: the oligosaccharide 3-Fucosyllactose (3FL), Lewis x trisaccharide (Lex), was configured to a concentration of 10mM with ultrapure water;

[0113] Reaction system: phosphate buffer 8ul, oligosaccharide preservation solution 1ul, enzyme 1ul, a total of 10ul reaction system, 37 ° C for 1 hour;

[0114] Detection of enzyme digestion reaction: two methods are used in this embodiment to detect the products after enzyme digestion of oligosaccharides:

[0115] Method 1: detect according to the L-fucose detection kit (megazyme, Ireland), the specific method can be found in the kit manual, the kit uses fucose dehydrogenase to oxidize fucose, and NADP+ to generate NADPH NADPH can be measured by the absorbance value at 340nm and quantified according to the calibration curve, so as to quantify the fucose produced by enzymatic cleavage of oligosaccharides;

[0116] Method 2: Detect accordin...

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Abstract

The invention belongs to the field of sugar biology technology and engineering, and particularly relates to a novel fucosidase of core fucosidase (Cfus) as well as enzymatic activity and application thereof. An experiment provided by the invention proves that the Cfus can excise core alpha1,3 fucose on an N-glycoprotein sugar chain, which is obviously different from the fucosidase which has no enzyme digestion activity on the glycoprotein core alpha1,3 fucose in the prior art; and at the same time, the Cfus can hydrolyze fucose of other fucose conjugates. The invention provides a new tool enzyme for the research of sugar biology and biomedicine; the tool enzyme may become a potential tool for allergy treatment; and the fucosidase can be used for structural analysis of N-glycoprotein sugarchains and functional research of core fucosylated sugar chains.

Description

technical field [0001] The invention belongs to the technical field of glycobiology and relates to molecular biology, biochemistry and pathogenic biology, in particular to fucosidase and its preparation method and application, especially a core fucosidase and its preparation method and application. Background technique [0002] The prior art discloses that glycosylation is one of the most important modifications in protein post-translational modification, and plays a very important role in the process of protein translation regulation and protein degradation, while fucosylation modification widely exists in various proteins. And oligosaccharides include N-oligosaccharides and O-oligosaccharides. [0003] Studies have shown that L-fucose is widely found in mammalian, plant and insect cells, and glycoconjugates containing L-fucose play a vital role in many physiological and pathological processes of these organisms, such as Inflammatory response, bacterial and viral infection...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N15/63
CPCC12N9/2402C12N9/2451C12Y302/01051
Inventor 陈力李天胜李梦洁
Owner FUDAN UNIV
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