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Method for immobilizing enzyme with aldehyde Arabic gum modified mesoporous silica

A technology of mesoporous silica and immobilized enzyme, applied in the field of detection, can solve the problems of toxicity to human body and ecological environment, large loss of glutaraldehyde activity, loss of penicillin acyltransferase activity, etc., and achieves high safety, organic solvent Tolerance-enhancing effect

Active Publication Date: 2020-07-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, glutaraldehyde is not suitable for all enzyme molecules. Some enzyme molecules, especially those with non-aqueous catalytic properties, lose their activity when exposed to glutaraldehyde. For example, Mateo et al. used glutaraldehyde to immobilize penicillin However, the activity of penicillin acylase after immobilization with glutaraldehyde is almost completely lost (see references for details: Mateo C, Palomo J M, Van Langen L M, et al.A new, mild cross-linkingmethodology to prepare cross-linked enzyme aggregates.[J].Biotechnology and Bioengineering,2004,86(3):273-276.), which greatly limits the scope of application of the covalent binding method
In addition, glutaraldehyde is toxic to the human body and the ecological environment, which also greatly limits the application range of the covalent bonding method.

Method used

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  • Method for immobilizing enzyme with aldehyde Arabic gum modified mesoporous silica
  • Method for immobilizing enzyme with aldehyde Arabic gum modified mesoporous silica
  • Method for immobilizing enzyme with aldehyde Arabic gum modified mesoporous silica

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preparation example Construction

[0073] The preparation method involved in the following examples is as follows:

[0074] The preparation method of mesoporous silica is as follows (mesoporous silica can also be purchased directly):

[0075] Specific steps are as follows:

[0076] Get 4g polyethylene oxide-propylene oxide-ethylene oxide triblock copolymer (P123) and add to 75mL concentration be that in the hydrochloric acid solution of 1.6mM, stir until polyethylene oxide-propylene oxide-epoxy The ethane triblock copolymer (P123) was completely dissolved to obtain the mixed solution 1; take 3.4mL 1,3,5-trimethylbenzene and add it to the mixed solution 1, and stir vigorously at 40°C for 4 hours to obtain the mixed solution 2; take 9.2 Add 5 mL of tetraethyl orthosilicate to the mixed solution 2, stir for 5 minutes and let it stand for 20 hours to obtain the mixed solution 3; add 5 mL of ammonium fluoride solution with a concentration of 9 mg / mL to the mixed solution 3 to obtain the mixed solution 4; The mixed...

Embodiment 1

[0080] Embodiment 1: the preparation of immobilized enzyme carrier MCFs-NH2

[0081] Specific steps are as follows:

[0082] Take 5g of gum arabic and add it to 15mL of sodium periodate solution (concentration: 100mM), react at 37°C and 200r / min for 3h to obtain reaction solution A; put reaction solution A in a dialysis bag at room temperature (25°C) Under treatment for 24 hours, the aldehyde-based gum arabic solution was obtained (the content of the aldehyde group in the aldehyde-based gum arabic solution was 83mM), and the aldehyde-based gum arabic solution needed to be placed in a refrigerator at 4°C for subsequent use (the preparation process of the aldehyde-based gum arabic solution can be seen figure 1 ); Take 1g of mesoporous silica (MCFs) and place it in an oven at 120°C for dehydration for 6h to obtain dehydrated mesoporous silica (MCFs); mix 1mL of 3-(aminopropyl)triethoxysilane and 50mL of toluene , to obtain a mixed solution; after mixing the dehydrated mesoporous...

Embodiment 2

[0085] Embodiment 2: the immobilization of enzyme (taking lipase CALB as example)

[0086] The free lipase and the immobilized lipase prepared by using glutaraldehyde as a coupling agent are used as a contrast, wherein the immobilized lipase prepared by using glutaraldehyde as a coupling agent is denoted as MCFs-NH2-G-CALB ( Method for preparing immobilized lipase using glutaraldehyde as coupling agent Reference: Gao J, Feng K, Li H, et al. Immobilized lipaseon porous ceramic monoliths for the production of sugar-derived oil gellingagent[J].RSC Advances , 2015, 5(84):68601-68609.).

[0087] After adding the lipase solution to the immobilized enzyme carrier prepared in Example 1 until the concentration of lipase in the mixture is 1.5 mg / mL (the concentration of lipase is detected by the Coomassie Brilliant Blue method), at 25 ° C, 200 r / min React for 1.5h to obtain a reaction solution; centrifuge the reaction solution to obtain a precipitate; wash the precipitate with phosphat...

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Abstract

The present invention discloses a method for immobilizing an enzyme with aldehyde Arabic gum modified mesoporous silica, and belongs to the technical field of detection. The invention provides an immobilized enzyme carrier, and the immobilized enzyme carrier can be used for the enzyme with the non-aqueous catalysis performance such as immobilized lipase; and compared with soluble lipase and the immobilized lipase prepared from glutaraldehyde as a coupling agent, the ester synthesis activity, the thermal stability and the organic solvent tolerance of the lipase immobilized with the immobilizedenzyme carrier are remarkably improved.

Description

technical field [0001] The invention relates to a method for immobilizing enzymes with mesoporous silicon oxide modified with aldehyde-based gum arabic, and belongs to the technical field of detection. Background technique [0002] Immobilized enzyme refers to an enzyme that plays a catalytic role in a certain space and can be used repeatedly and continuously. Compared with free enzymes, immobilized enzymes overcome the shortcomings of free enzymes while maintaining their high-efficiency, specificity and mild enzymatic reaction characteristics. They have high storage stability, easy separation and recovery, and can be reused many times. , continuous controllable operation, simple process and a series of advantages. At present, immobilized enzymes are widely used in chemistry, biology, bioengineering, medicine, life science and other disciplines. [0003] Immobilization of enzymes (Immobilization of enzymes) is a type of technology that binds or confines enzymes in a certai...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12N11/10C12N11/089C08B37/00
CPCC12N11/14C12N11/10C12N11/08C12N9/20C12Y301/01003C08B37/0087Y02P20/584
Inventor 喻晓蔚徐岩靳文斌
Owner JIANGNAN UNIV
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