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Immobilized enzyme based protein enzymolysis method

An immobilized enzyme and protein enzyme technology, applied in the field of protein enzymatic hydrolysis and protein enzymatic hydrolysis based on immobilized enzyme, can solve the problems of enrichment of protein samples to be tested, affecting the efficiency and accuracy of enzymatic hydrolysis, and improve the detection rate. , the effect of improving efficiency and fast sedimentation rate

Inactive Publication Date: 2018-01-16
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This technology uses tiny microscopic spherules that slowly sink down into water during their production or storage period. These small balls are made up mostly of carboxylate groups with different types of amino acids attached through an ester bond between them. They have many advantages when used for this purpose: they don't require strong chemical reagents like acid) but still work well even at room temperatures (which makes analysis easier). When placed together under pressure, these minute objects break open easily allowing liquid to flow out without leaving any residue behind. By measuring how much material remains after each treatment, researchers may determine if certain substances were contained within specific samples being analyzed.

Problems solved by technology

Technological Problem addressed in this patented text relates to improving the effectiveness of enzylical chemistry methods that involve chemical reactions between proteins or other molecules under specific conditions. These techniques require high temperatures and are slow because they rely heavily upon water solubility. To solve these issues, an improved approach called Immunolocalization was developed where certain carriers were added into the reaction mixture beforehand with the targeted substances being analyzed.

Method used

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  • Immobilized enzyme based protein enzymolysis method
  • Immobilized enzyme based protein enzymolysis method
  • Immobilized enzyme based protein enzymolysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0109] A protein enzymatic hydrolysis method based on immobilized enzymes, the specific operation steps are as follows:

[0110] (1) Preparation of thermosensitive immobilized trypsin microspheres

[0111] (a) Preparation of carrier material

[0112] The carrier material is formed by copolymerization of N-isopropylacrylamide and acrylic acid at a mass ratio of 9:1. The specific preparation method is:

[0113] Preparation of copolymerized microspheres by rapid membrane emulsification method: Add acrylic acid, N-isopropylacrylamide, initiator, and crosslinking agent to deionized water, N-isopropylacrylamide, initiator, crosslinking agent, and deionized water The mass ratio is 10:0.2:1.5:500, stirred until the solid is completely dissolved, and prepared into an aqueous phase; mix Span-80 and cyclohexane at a mass ratio of 1:20, stir at room temperature until Span-80 is completely dissolved, Prepare the oil phase; mix the oil phase and the water phase at a volume ratio of 1:5, s...

Embodiment 2

[0124] A kind of protein enzymolysis method based on immobilized enzyme, its specific operation steps are as follows:

[0125] (1) Preparation of thermosensitive immobilized trypsin microspheres

[0126] (a) Preparation of carrier material

[0127] The carrier material is formed by copolymerization of N-isopropylacrylamide and methacrylic acid at a mass ratio of 1:1. The specific preparation method is:

[0128] Copolymerized microspheres were prepared by soap-free emulsion polymerization: the monomer N-isopropylacrylamide, methacrylic acid and cross-linking agent were dissolved in 200mL deionized water and put into a 250mL four-necked flask, N-isopropylacrylamide The mass ratio of acrylamide, cross-linking agent and deionized water is 100:0.1:20000. Nitrogen gas is introduced and mechanical stirring is carried out. After 15 minutes of stabilization, the initiator is added, and the temperature is raised to 80°C, and the reaction is continued for 6 hours. After the reaction, t...

Embodiment 3

[0135] A kind of protein enzymolysis method based on immobilized enzyme, its specific operation steps are as follows:

[0136] (1) Preparation of thermosensitive immobilized chymotrypsin microspheres

[0137] (a) Preparation of carrier material

[0138] The carrier material is formed by copolymerization of N-isopropylacrylamide and acrylic acid at a mass ratio of 20:1. The specific preparation method is:

[0139] Copolymerized microspheres were prepared by precipitation polymerization: acrylic acid, N-isopropylacrylamide, initiator and crosslinking agent solvent were dissolved in 80mL aqueous solution, and the concentration of N-isopropylacrylamide was 7g / L; N in the solution - The mass ratio of isopropylacrylamide, initiator and crosslinking agent is 10:1:0.5; nitrogen is passed through the solution for 30 minutes, and then the above solution is heated to 70°C in an oil bath or water bath for 8 hours of reaction; After the end, the temperature of the reaction solution was l...

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Abstract

The invention relates to an immobilized enzyme based protein enzymolysis method. The method adopts temperature sensitive microspheres as the immobilized enzyme carrier, performs temperature regulationof the hydrophilic and hydrophobic properties of the temperature sensitive immobilized enzyme microspheres so as to control the protein enzymolysis process, i.e. when the enzymolysis temperature is higher than a critical phase inversion temperature of the temperature sensitive immobilized enzyme microspheres, the microspheres are hydrophobic, while a to-be-tested protein sample is enriched, enzymolysis is carried out on the microspheres, when the enzymolysis temperature is lower than the critical phase inversion temperature of the temperature sensitive immobilized enzyme microspheres, enzymolysis is stopped and peptide fragment is released, thereby improving the enzymolysis efficiency and detection rate of the sample peptide fragment. The protein enzymolysis method provided by the invention has the characteristics of simple steps, convenient operation and the like, and is conducive to research of proteomics and related fields.

Description

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Claims

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Application Information

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Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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