Immobilized enzyme reactor based on trimethylolpropane trimethacrylate monolithic column

A technology of trimethylolpropane trimethacrylate and immobilized enzyme, which is applied in the directions of enzyme production/bioreactor, bioreactor/fermenter for specific purposes, bioreactor/fermenter combination, etc. Simple, stable and reproducible, efficient and rapid enzymatic hydrolysis

Active Publication Date: 2019-01-25
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional organic monolithic columns have no residual double b

Method used

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  • Immobilized enzyme reactor based on trimethylolpropane trimethacrylate monolithic column
  • Immobilized enzyme reactor based on trimethylolpropane trimethacrylate monolithic column
  • Immobilized enzyme reactor based on trimethylolpropane trimethacrylate monolithic column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Preparation of organic monolithic column: 200 μL of TRIM, 680 μL of isooctane and 120 μL of toluene as porogen, 3.6 mg of azobisisobutyronitrile as initiator, mixed with ultrasound (150 W, 10 min) to form a pre-polymerization solution Inject into the treated capillary, seal both ends of the capillary with rubber stoppers, place in a 50°C water bath, take out the capillary after reacting for 24 h, and rinse with acetonitrile to remove unreacted solution and initiator.

[0019] Reduction of trypsin disulfide bond: use 20 mM Tris-HCl buffer (pH 8.0) to prepare trypsin solution with a concentration of 10 mg / mL and tris(2-carbonylethyl) phosphate hydrochloride with a concentration of 1 mg / mL (TCEP) solution. Then the two solutions were mixed according to the ratio of TCEP:trypsin solution at 1:10 (v / v), and the mixed solution was reacted at 25°C for 3 h to reduce and break the disulfide bond in trypsin. Then the mixture was centrifuged at 4°C (10000 rpm) for 10 min to remov...

Embodiment 2

[0022] Enzyme activity assay: Trypsin activity assay is achieved by enzymatically hydrolyzing BAEE into BA. Using 5-25mM BAEE (in 20 mM Tris-HCl buffer, pH 8.0) as the substrate, it was digested by the same monolithic column under the same conditions, and the flow rate was 1 μL / min. The enzymatic hydrolysis products were collected by EP tubes and detected by reversed-phase high performance liquid chromatography. Michaelis constant (K m ) and maximum response rate (V max ) is calculated by the Mie equation. Use the Linewaver-Burk plot to plot 1 / V versus 1 / [S]:

[0023]

[0024] A straight line can be obtained. The intercept of the straight line on the horizontal axis is -1 / Km, and the vertical intercept is 1 / V max , can find K m with V max .

[0025] The detection conditions of high performance liquid chromatography are as follows:

[0026] Mobile phase: 80mM KH 2 PO 4 (pH 3.87):methanol=7:3

[0027] Flow rate: 1ml / min

[0028] Column temperature: 25°C

[0029]...

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Abstract

The invention relates to an immobilized enzyme reactor based on a trimethylolpropane trimethacrylate monolithic column. Trimethylolpropane trimethacrylate (TRIM) having three double bonds, toluene, isooctane and azobisisobutyronitrile were used as fixed matrix for trypsin immobilization. There were some remaining double bonds on the surface of TRIM skeleton, which could react with free sulfhydrylgroups on the surface of trypsin at room temperature and bind the trypsin to the monolithic column. The enzyme reactor prepared by the invention does not need to introduce a functional group on the fixed substrate, and the preparation is more convenient; but also avoids trypsin functionalization and enzyme denaturation and inactivation. This study provides a new way to study the immobilized substrate of enzyme reactor. The enzyme reactor can be used for high efficiency and rapid enzymatic hydrolysis of proteins.

Description

technical field [0001] The invention relates to an immobilized enzyme reactor with trimethylolpropane trimethacrylate monolithic column as the matrix, and trimethylolpropane trimethacrylate (TRIM) is prepared by "mercapto-ene" click reaction The invention discloses an immobilized enzyme reactor with an organic monolithic column as a substrate, belonging to the technical field of enzyme reactors, and aims to study a new type of enzyme immobilized substrate, and the prepared enzyme reactor is used for efficient and rapid enzymolysis of protein samples. Background technique [0002] Hydrolysis of proteins into peptides is a key step in "bottom-up" proteomics research. However, conventional solution enzymatic hydrolysis has disadvantages such as long time-consuming, low activity, self-dissolution of protease, non-reusable protease, etc. Therefore, immobilization of enzyme is an effective method to solve the above problems. Enzyme immobilization has the following advantages: sho...

Claims

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Application Information

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IPC IPC(8): C12N11/08C12M1/40C12M1/24
CPCC12M21/18C12M23/16C12N9/6427C12N11/08C12Y304/21004
Inventor 黄艳萍范培茹刘照胜
Owner TIANJIN MEDICAL UNIV
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