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A kind of method using aldehyde-based gum arabic modified mesoporous silica to immobilize enzyme

A technology of mesoporous silica and immobilized enzymes, applied in the field of detection, can solve the problems of toxicity to human body and ecological environment, loss of penicillin acyltransferase activity, large loss of glutaraldehyde activity, etc., and improve the tolerance of organic solvents. Effect

Active Publication Date: 2022-07-05
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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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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  • A kind of method using aldehyde-based gum arabic modified mesoporous silica to immobilize enzyme
  • A kind of method using aldehyde-based gum arabic modified mesoporous silica to immobilize enzyme
  • A kind of method using aldehyde-based gum arabic modified mesoporous silica to immobilize enzyme

Examples

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preparation example Construction

[0073] The preparation methods involved in the following examples are 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] Take 4g of polyethylene oxide-propylene oxide-ethylene oxide triblock copolymer (P123) and add it to 75mL of hydrochloric acid solution with a concentration of 1.6mM and stir until polyethylene oxide-propylene oxide-epoxide Ethane triblock copolymer (P123) was completely dissolved to obtain mixed solution 1; 3.4 mL of 1,3,5-trimethylbenzene was added to mixed solution 1, and vigorously stirred at 40 °C for 4 h to obtain mixed solution 2; Add mL tetraethyl orthosilicate to mixed solution 2, stir for 5 min and then let stand for 20 h to obtain mixed solution 3; take 5 mL of ammonium fluoride solution with a concentration of 9 mg / mL and add it to mixed solution 3 to obtain mixed solution 4; The mixed solution 4 was placed in a cryst...

Embodiment 1

[0080] Example 1: Preparation of immobilized enzyme carrier MCFs-NH2

[0081] Specific steps are as follows:

[0082] Add 5 g of gum arabic to 15 mL of sodium periodate solution (concentration of 100 mM), and react at 37 °C and 200 r / min for 3 h to obtain reaction solution A; put reaction solution A in a dialysis bag at room temperature (25 °C) Under treatment for 24h, the aldehyde-based gum arabic solution was obtained (the content of aldehyde-based gum arabic in the aldehyde-based gum arabic solution was 83 mM). figure 1 ); take 1 g of mesoporous silica (MCFs) and dehydrate it in an oven at 120 °C for 6 h to obtain dehydrated mesoporous silica (MCFs); mix 1 mL of 3-(aminopropyl)triethoxysilane with 50 mL of toluene , to obtain a mixed solution; after mixing the dehydrated mesoporous silica and the mixed solution, react at 50 °C for 6 h to obtain a reaction solution B; filter the reaction solution B to obtain a precipitate A; wash the precipitate A with ethanol for many time...

Embodiment 2

[0085] Example 2: Immobilization of enzymes (taking lipase CALB as an example)

[0086] The free lipase and the immobilized lipase prepared by using glutaraldehyde as the coupling agent were used as controls, and the immobilized lipase prepared by using glutaraldehyde as the coupling agent was recorded 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 the lipase solution added to the immobilized enzyme carrier prepared in Example 1 reached a concentration of 1.5 mg / mL of lipase in the mixed solution (the lipase concentration was detected by the Coomassie brilliant blue method), at 25° C., 200 r / min The reaction was performed for 1.5 h to obtain a reaction solution; the reaction solution was centrifuged to obtain a precipitate; the pr...

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Abstract

The invention discloses a method for immobilizing an enzyme with mesoporous silica modified by aldehyde-based gum arabic, and belongs to the technical field of detection. The present invention provides an immobilized enzyme carrier, which can be used for immobilizing lipase and other enzymes with non-aqueous phase catalytic properties; the immobilized enzyme carrier prepared with free lipase and using glutaraldehyde as a coupling agent Compared with lipase, the ester synthesis activity, thermal stability and organic solvent tolerance of the lipase immobilized using the immobilized enzyme carrier of the present invention are all significantly improved.

Description

technical field [0001] The invention relates to a method for using aldehyde-based gum arabic modified mesoporous silica to immobilize an enzyme, and belongs to the technical field of detection. Background technique [0002] Immobilized enzyme refers to an enzyme that plays a catalytic role within a certain spatial range and can be used repeatedly and continuously. Compared with the free enzyme, the immobilized enzyme overcomes the shortcomings of the free enzyme while maintaining its high efficiency, specificity and mild enzyme catalytic reaction characteristics, showing high storage stability, easy separation and recovery, and can be reused many times. , continuous and controllable operation, simple process and a series of advantages. At present, immobilized enzymes are widely used in the fields of chemistry, biology, bioengineering, medicine and life sciences. [0003] Immobilization of enzymes is a kind of technology that binds or confines enzymes in a certain area with...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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