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Low Residue Inorganic-Organic Hybrid Monolithic Matrix Immobilized Enzyme Reactor and Its Preparation

A technology of immobilized enzymes and organic hybridization, which is applied in the direction of immobilization on/in the organic carrier to achieve good preparation reproducibility, mild preparation conditions, and high-throughput protein enzymatic hydrolysis

Active Publication Date: 2016-09-28
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the silanol groups on the surface of hybrid silica monolithic columns are suppressed to a certain extent compared with silica monolithic columns, the non-specific adsorption of proteins cannot be completely avoided.

Method used

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  • Low Residue Inorganic-Organic Hybrid Monolithic Matrix Immobilized Enzyme Reactor and Its Preparation
  • Low Residue Inorganic-Organic Hybrid Monolithic Matrix Immobilized Enzyme Reactor and Its Preparation
  • Low Residue Inorganic-Organic Hybrid Monolithic Matrix Immobilized Enzyme Reactor and Its Preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Preparation of hybrid silica gel monolithic matrix material: Weigh 540mg of PEG-10000, dissolve it fully with 5mL of 0.01M acetic acid, then add 1.8mL of tetramethoxysilane and 0.6mL of vinyltrimethoxysilane, and stir for 1h at 0°C , to make reaction solution A. Weigh 2 mg of ammonium persulfate, dissolve it in 200 μL of reaction solution A, then add 200 μL of absolute ethanol and 10 μL of methacrylic acid in sequence, and shake and sonicate for 5 minutes. After cooling, add 50 μL of water and 50 μL of 0.5M ammonia water, pour into a capillary after shaking, and put it in a 40°C water bath for 24 hours.

[0026] 2. Substrate surface modification: After taking out the monolithic column, wash the pore-forming agents such as PEG-10000 and other unreacted small molecules with water. Weigh 10 mg and 20 mg of EDC and NHS, dissolve them in 1 mL of 50 mM pH 5.0 phosphate buffered saline, and pass them through the monolithic column for 4 hours, then rinse the unreacted EDC a...

Embodiment 2

[0029] Investigation of the recovery rate of proteins and peptides on the hybrid silica monolithic material matrix modified with PEI: prepare 1 mg / mL each of peptide (DRVYHPHL) and BSA, dilute with 50 mM ammonium bicarbonate (pH8.0) respectively, and mix at a ratio of 1:1 , the concentration of the mixed solution is 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, and draw the standard curve of BSA (2a) and polypeptide (2b), the separation conditions are as follows: chromatographic column: Proteonavi column (4.6mm i.d×150mm, 5μ, ), mobile phase: A: 0.1%TFA+H 2 O;B: 0.1%TFA+ACN; Gradient: 0-5min, 5%B, 5-35min, 5%B-80%B; flow rate: 0.5mL / min, detection: 214nm; injection volume: 20μL. In order to investigate the adsorption of the matrix to the sample, a section of 5 cm long monolithic column was intercepted, and 100 μg / mL mixed sample was passed through at 1 μL / min, collected for 100 min, and then 20 μL was injected, and the chromatographic picture Such as figure 1 As s...

Embodiment 3

[0031] Evaluation of low-residue hybrid silica monolithic matrix enzyme reactor: 0.1mg / mL BSA and 0.05mg / mL myoglobin (Myo) were continuously passed into the immobilized enzyme reactor at 1μL / min (0.25mm i.d×50mm) In the process, the components were collected and detected by MALDI-TOF MS. After searching the database, the sequence coverage of BSA was 75% (such as figure 2 a), the sequence coverage of myoglobin is 98% (eg figure 2 b).

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Abstract

The present invention relates to a low residue inorganic-organic hybrid silica gel whole matrix immobilized enzyme reactor. According to the present invention, tetramethoxy silane, vinyl trimethoxy silane and methacrylic acid are adopted as monomers, polyethylene glycol is adopted as a pore-forming agent, in-situ polymerization is performed under an ethanol-ammonia water mixed system to obtain a hybrid rigid matrix having a porous structure, and a hydrophilic active reaction group is introduced on the surface of the matrix, such that protein or protease molecules are firmly bonded on the whole matrix so as to prepare the high activity and low residue immobilized enzyme reactor; the method has advantages of high stability, simpleness, rapidness and the like; and compared with the conventional protein immobilization method, the method of the present invention has the following characteristics that: the obtained whole matrix has the high activity reaction group and can quickly react with protein and other molecules having the primary amine group, and the recovery rate of proteins or enzymolysis products is significantly increased due to introduction of the hydrophilic group on the surface of the matrix.

Description

technical field [0001] The invention relates to a low-residue inorganic-organic hybrid integral substrate immobilized enzyme reactor, which can be used for low-residue and rapid enzymolysis of proteins. Background technique [0002] In proteomic analysis methods based on peptide fingerprinting and MS / MS peptide sequencing, protein enzymatic digestion is a very important sample pretreatment step. However, the commonly used free solution enzymatic hydrolysis method has many disadvantages such as long enzymatic hydrolysis time, unrecyclable enzymes, and easy self-degradation. In recent years, in order to overcome these problems, people have immobilized proteases on different substrates to prepare immobilized enzyme reactors (IMER, immobilized enzymatic reactor), thereby improving the efficiency of enzymatic hydrolysis of proteins, avoiding the manual operation of protein samples, and reducing the Possibility of protein sample contamination. At present, a variety of materials ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08C08G81/02C08F220/06C08F230/08C08F8/00
Inventor 张丽华袁辉明陈玲凡杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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