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Immobilized glycosidase reagent and preparing method thereof

A technology of glycosidase and reagent, which is applied in the direction of fixing on or in the inorganic carrier, fixed on/in the organic carrier, etc., can solve the problems of limiting enzymatic hydrolysis efficiency, so as to improve the efficiency of enzymatic hydrolysis and sample processing Efficiency and simplification of operation steps

Inactive Publication Date: 2015-11-11
BEIJING PROTEOME RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many immobilized enzyme carrier materials have been developed, such as magnetic nanoparticles, graphene oxide, polymer membranes, mesoporous materials, etc., which greatly shorten the reaction time required for enzymatic hydrolysis, but enzymatic hydrolysis based on these immobilized enzyme technologies The reactions are all carried out under complex phase conditions, which limits the improvement of enzymatic hydrolysis efficiency

Method used

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  • Immobilized glycosidase reagent and preparing method thereof
  • Immobilized glycosidase reagent and preparing method thereof
  • Immobilized glycosidase reagent and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A kind of preparation method of immobilized glycosidase reagent, concrete steps are as follows:

[0052] (1) The preparation method of the initiator of surface-initiated atom transfer radical polymerization: the silane coupling agent and the protonic acid scavenger are reacted in an ice bath, and then the initiator of atom transfer radical polymerization is added, and the reaction is carried out to obtain , wherein the molar ratio of silane coupling agent, atom transfer radical polymerization initiator and protonic acid scavenger is 0.8:1:1.

[0053] (2) Preparation method of magnetic nanoparticles with surface-initiated atom transfer radical polymerization initiator: the magnetic nanoparticles are modified by oleic acid coating, and the surface-initiated atom transfer radical polymerization initiator is dissolved in a solvent In the process of mixing, a ligand exchange reaction occurs to generate a coordination bond, that is, the solvent is benzene or toluene-n-hexane,...

Embodiment 2

[0058] Preparation of thermosensitive magnetic fluid carrier immobilized peptides by a SI-ATRP method N - Glycosidase F and thermosensitive magnetic fluid carrier immobilized peptide N The method for enzymolysis of glycoprotein by glycosidase F, the specific steps are as follows:

[0059] (1) Synthesis of SI-ATRP initiator: SI-ATRP initiator was synthesized by reacting 3-aminopropyltriethoxysilane with 2-bromoisobutyryl bromide. A silane coupling agent for ligand exchange reaction of iron atoms in magnetic nanoparticles, and ATRP initiator at the other end; the specific steps are as follows: 0.8 mmol of 3-aminopropyltriethoxysilane and 0.8 mmol of triethylamine are added Into 20ml of toluene, after mixing, introduce nitrogen to deoxygenate and ice bath for 30min to obtain silane coupling agent, then slowly add 0.8mmol of 2-bromoisobutyryl bromide dropwise to the mixture, stir vigorously in ice bath for 3h , slowly heated to room temperature and continued the reaction for 10h...

Embodiment 3

[0081] immobilized peptide N - Functional evaluation of glycosidase F reagent:

[0082] (1) Comparative enzymatic hydrolysis and enzymatic hydrolysis reaction kinetics of ribonuclease B:

[0083] First, using ribonuclease B, a molecular weight of approximately 15 kDa containing only a single N Glycoproteins with glycosylation sites were used as standard glycoproteins, and the immobilized peptides obtained were evaluated by comparing them with traditional solution enzymatic hydrolysis and this immobilized enzymatic hydrolysis. N - Function of the Glycosidase F reagent. The specific operation steps are as follows:

[0084] A. Prepare 10 μg / μL RNase B solution, add DTT, treat in a 95°C water bath for 10 minutes, then add IAA and place in the dark for 1 h for alkylation.

[0085] B. Take 4 μL of the denatured glycoprotein solution obtained in step 1 and add 1 mL of immobilized peptide N -Glycosidase F reagent, ultrasonically vibrated for 3 minutes at room temperature, heated ...

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Abstract

The invention discloses an immobilized glycosidase reagent. The immobilized glycosidase reagent comprises a thermo-sensitive magnetic fluid carrier material and glycosidase. The glycosidase is fixed to the thermo-sensitive magnetic fluid carrier material. The thermo-sensitive magnetic fluid carrier material comprises thermo-sensitive monomers, monomers with active functional groups capable of being combined with the glycosidase and magnetic nano-particles with an initiator for surface-initiated atom transfer radical polymerization. The thermo-sensitive monomers and the monomers with the active functional groups capable of being combined with the glycosidase are copolymerized into a polymer chain, the copolymerized polymer chain and the magnetic nano-particles are connected through coordinate bonds. The polymer chain and the glycosidase are connected through carbon-nitrogen bonds. By means of the immobilizated glycosidase reagent, the immobilized carrying amount of the glycosidase of unit mass immobilized glycosidase reagent and the stability of the immobilized glycosidase reagent are obviously improved, the enzymolysis efficiency is improved, the time needed by the thermo-sensitive magnetic fluid immobilized enzyme to complete glycoprotein enzymolysis is extremely short, the sample treating efficiency is obviously improved, the sample purity is improved, and the mass spectrum identification result is improved.

Description

technical field [0001] The present invention relates to a reagent, in particular to an immobilized glycosidase reagent and a preparation method thereof. Background technique [0002] As one of the most extensive, complex and important post-translational modifications, protein glycosylation has been shown to exist in more than half of mammalian cellular proteins, and it not only affects the spatial conformation, biological activity, transport and localization of proteins , and plays a crucial role in specific biological processes such as molecular recognition, cellular communication, and signal transmission. According to the different glycosylation sites, glycoproteins can be divided into three categories: N-glycoproteins, O-glycoproteins and GPI-anchored proteins, among which N-glycoproteins are the most widely distributed, and the lack of content or structure Abnormal changes in osteoarthritis, cystic fibrosis, cancer are closely related to a variety of diseases. Therefor...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/08
Inventor 徐福建郑斐钱小红秦伟捷
Owner BEIJING PROTEOME RES CENT
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