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Preparation of porous integral material immobilized enzyme micro-reactor

An integral material, microreactor technology, applied in the field of enzymatic hydrolysis of protein samples, to achieve the effects of shortened reaction time, good permeability and low cost

Active Publication Date: 2009-08-05
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

Sakai-Kato etc. have prepared a kind of silica gel integral material embedded type enzyme reactor by sol-gel method, can make 50mMBAEE enzymolysis (Sakai-Kato, K.; Kato, M.; Toyo'oka , T.Anal.Chem.2002, 74, 2943-2949), but this enzyme reactor can not effectively enzymolyze macromolecular proteins

Method used

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  • Preparation of porous integral material immobilized enzyme micro-reactor
  • Preparation of porous integral material immobilized enzyme micro-reactor
  • Preparation of porous integral material immobilized enzyme micro-reactor

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Experimental program
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Embodiment Construction

[0024] The organic-inorganic hybrid silica gel monolith immobilized enzyme reactor was prepared according to the following steps.

[0025] 1) Take a certain amount of TEOS, APTES, absolute ethanol, water and CTAB according to the ratios listed below and mix them thoroughly, and then introduce them into a pretreated capillary with an inner diameter of 10-300 μm through a syringe. The introduction process is after the raw materials are mixed. completed within 2 minutes. Seal with silicone rubber, and polymerize at 25-80°C for 12-24 hours; an organic-inorganic hybrid silica gel monolithic material carrier was prepared by sol-gel method and supramolecular template method; the specific parameters of the synthesis example are as follows 1 shown;

[0026] 2) Activation: The prepared monolithic material was first washed with ethanol to remove CTAB through a constant flow pump, and then washed with water and 100 mM phosphate buffer (pH 8.0) for 30 min. Then, 100 mM phosphate buffer (...

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Abstract

The invention relates to a zymohydrolysis method of protein samples, in particular to a porous organic-inorganic hybrid silica bulk material carrier which is prepared and obtained by using tetraethoxy silane and aminopropyl triethoxysilane as precursor molecules, using cetyltrimethylammonium trimethyl bromide ammonia as template molecules, using absolute ethyl alcohol as cosolvent through a sol-gel method and a template molecule self-assembly method. Hybrid silica bulk materials are activated by glutaric dialdehyde. After trypsinase is fixed, an organic-inorganic hybrid silica bulk material immobilized enzyme reactor is prepared. The invention has the advantages of simple and convenient preparation method, good permeability of the enzyme reactor, little counter pressure, high mechanical strength and high zymohydrolysis activity. Compared with free solution zymohydrolysis, the invention largely shortens reaction time, avoids the self-degradation of enzyme, can be repeatedly used, and also can be combined with an HPLC-MS / MS or CE-MS / MS system, thereby the on-line analysis of samples is realized.

Description

technical field [0001] The invention relates to an enzymolysis method for protein samples, in particular to an immobilized enzyme microreactor based on a porous monolithic material, which realizes efficient and rapid enzymolysis of protein samples. Background technique [0002] As an important branch of chemical enzyme engineering, enzyme immobilization refers to the immobilization of target enzyme molecules on specific carriers through adsorption, embedding, cross-linking, covalent bonding and other methods to exert the biocatalytic effect of enzymes. Compared with protein enzymatic hydrolysis reactions in solution, immobilized enzymes have the advantages of higher enzyme / substrate ratio, higher enzymatic hydrolysis efficiency, reusability, and reduced enzyme self-degradation. In addition, the use of immobilized enzyme reactors can avoid manual sample handling, thereby reducing the possibility of protein sample contamination. [0003] The performance of immobilized enzymes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/04C12N11/14
Inventor 张丽华马俊锋梁振张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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