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A kind of preparation method of metal wire immobilized enzyme reactor modified by atom transfer radical polymerization

An immobilized enzyme and atom transfer technology, applied in the field of biochemistry, can solve the problems of inconvenient operation, unfavorable control, and long enzymatic hydrolysis time, and achieve the effects of simple and convenient use, easy separation, and reduced enzymatic hydrolysis time.

Active Publication Date: 2016-08-17
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the shortcomings of the traditional solution enzymatic hydrolysis method, such as long enzymatic hydrolysis time, interference of enzyme self-cutting substances, and inability to reuse proteases, etc., in order to solve the problems existing in the solution enzymatic hydrolysis process, a fixed enzyme was introduced and developed in proteome research. enzyme technology
There have been many reports on immobilized enzyme reactors, and their materials are also diverse, including gold nanoparticles, magnetic nanoparticles coated with silica, graphene oxide, chip materials, cotton yarns, mesoporous materials, and quartz. Capillary monolithic column, hybrid monolithic column immobilized enzyme reactor, etc., but the operation is still not convenient enough
[0003] Atom transfer radical polymerization (ATRP) is a controllable radical polymerization method first proposed by Professor Matyjaszewki K.’s group in 1995, but due to the catalytic O in the traditional ATRP reaction 2 agent pair H 2 O is more sensitive, which is not conducive to the control of the reaction. Professor Matyjaszewki K.'s group further developed the method of electron transfer to generate catalyst atom transfer radical polymerization (AGET ATRP), which effectively overcomes this problem

Method used

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  • A kind of preparation method of metal wire immobilized enzyme reactor modified by atom transfer radical polymerization
  • A kind of preparation method of metal wire immobilized enzyme reactor modified by atom transfer radical polymerization
  • A kind of preparation method of metal wire immobilized enzyme reactor modified by atom transfer radical polymerization

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1, Preparation of Wire-immobilized Enzyme Reactor Based on Atom Transfer Radical Polymerization Modification

[0049] This example will describe in detail the preparation method of the wire-immobilized enzyme reactor based on atom transfer radical polymerization modification, and its preparation flow chart is as follows figure 1 shown.

[0050] 1. Synthesis of Atom Transfer Radical Polymerization Initiator Containing Mercapto

[0051] Using 11-mercapto-1-undecyl alcohol and 2-bromoisobutyryl bromide to synthesize an initiator with a mercapto group at one end and an atom transfer radical polymerization (ATRP) initiation group at the other end. The details are as follows: Dissolve 0.3 g of 11-mercapto-1-undecanol in 6 mL of tetrahydrofuran (providing a liquid environment for the reaction), then add 108 μL of pyridine (catalyst), mix and pass through nitrogen to remove oxygen, and at the same time After bathing for 30 minutes, slowly add 164 μL of 2-bromoisobutyr...

Embodiment 2

[0067] The determination of the enzymolysis peptide segment coverage rate of the immobilized enzyme reactor prepared in embodiment 2, embodiment 1

[0068] In this example, the wire-immobilized enzyme reactor prepared in Example 1 was used to perform enzymatic hydrolysis and traditional solution enzymatic hydrolysis, and the enzymatic hydrolysis peptide coverage of bovine serum albumin (BSA) was measured by these two methods. And the results were compared and analyzed. details as follows:

[0069] Prepare a bovine serum albumin (BSA) solution with a final concentration of 2 mg / mL (the solvent is 50 mM Tris-HCl buffer solution, and the formula is the same as that described in step 7 of Example 1), and dithiothreitol (DTT) is added to make DTT The final concentration of IAA is 10mM, then placed in boiling water to heat and denature for 10min, after cooling to room temperature, add iodoacetamide (IAA) to make the final concentration of IAA 50mM, place in the dark for 1h, add DTT...

Embodiment 3

[0072] The immobilized enzyme reactor enzymolysis efficiency stability measurement that embodiment 3, embodiment 1 prepare

[0073] In this example, bovine serum albumin (BSA) was used as the protein sample to be hydrolyzed, and the stability of the enzymatic hydrolysis efficiency of the wire-immobilized enzyme reactor prepared in Example 1 was measured. details as follows:

[0074] Using the same wire-immobilized enzyme reactor prepared in Example 1, 8 parts of BSA were enzymatically hydrolyzed within one month (on Monday and Thursday every week), the specific enzymatic hydrolysis methods and steps are as in the example as described in 2. The products after 8 times of BSA hydrolysis were analyzed by MALDI-TOF MS mass spectrometry. Mass spectrometry conditions are as described in Example 2. The resulting mass spectral data were submitted to MASCOT for peptide mass fingerprinting analysis. Peptide mass fingerprint analysis was performed, and the resulting amino acid sequenc...

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Abstract

The present invention discloses a preparation method for an immobilized enzyme reactor. The method comprises: 1) preparing an initiator having a mercapto terminal and an atom transfer radical polymerization initiating group terminal; 2) adopting mercapto to immobilize the initiator on the surface of a metal wire; 3) adopting an atom transfer radical polymerization reaction of a glycidyl methacrylate monomer to immobilize the epoxy group on the surface of the metal wire; 4) adopting ethylenediamine to make the epoxy group be subjected to ring opening and expose the amino group; 5) adopting a reaction of the amino group and glutaraldehyde to modify the aldehyde group on the surface of the metal wire; 6) adopting a reaction of the aldehyde group and the amino group of protease to immobilized the protease on the surface of the metal wire; and 7) adopting an amino-containing compound to block the residual aldehyde group to obtain the metal wire immobilized enzyme reactor. The immobilized enzyme reactor has characteristics of simple and convenient use, easy separation from the solution, and recycling, and has a significantly reduced enzymolysis time compared with the conventional solution enzymolysis method.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to a preparation method of a metal wire immobilized enzyme reactor modified by atom transfer radical polymerization. Background technique [0002] The two main research strategies in proteomics research are "top-down" strategy and "bottom-up" strategy. Since the "top-down" strategy can only be applied to a single protein or a simple protein mixture, the most commonly used strategy is still the "bottom-up" research strategy. In view of the fact that in the two main workflows of "bottom-up" (peptide mass fingerprinting and peptide sequence labeling), it is necessary to perform mass spectrometry (MS) analysis on the enzymatic hydrolyzate of the protein, and it has become a key factor in the identification and identification of proteins. The most commonly used method for the study of its post-translational modification. Therefore, enzymatic digestion of proteins plays a key role in the study...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/14C12N11/08C08F292/00C08F220/32C08F8/32
Inventor 钱小红张养军周廉淇
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA