Method for immobilizing pseudomonas fluorescens lipase by metal organic framework

A technology of immobilizing enzymes and lipases, which is applied in the field of bioengineering, can solve the problems of low catalytic activity of lipases, difficulty in separating and reusing, and affecting catalytic activity, so as to maintain catalytic activity and enantioselectivity, repeat The effect of multiple uses and high selectivity

Inactive Publication Date: 2020-07-31
HUNAN INSTITUTE OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme preparations are generally added to the enzyme-catalyzed resolution reaction in the form of enzyme powder. Once this exogenous enzyme is added, it is difficult to separate and reuse it. Because the price of enzyme preparations is generally relatively high, the cost of a single use will be high
In addition, the catalytic activity of lipase in the aqueous reaction system is not high, and it is prone to agglomeration in the organic system, which affects the catalytic activity.
These unfavorable factors of enzyme preparation have caused its application to be affected to a certain extent

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Put 50 mg of Uio-66(Zr) in the reaction tube, and then add 200 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at a concentration of 10 mg / mL , stirred and activated at 25°C for 1.5 h, and activated Uio-66(Zr) was initially obtained; then added 200 μL of phosphate buffer solution containing N-hydroxysuccinimide at a concentration of 12 mg / mL, and reacted at 25°C 1.5 h; Finally, add 3.6 mL of Pseudomonas fluorescens lipase solution containing 35 mg to the above-mentioned mixture containing activated Uio-66(Zr), cross-link at a certain temperature for 4-20 h, and centrifuge , washed with deionized water, and then freeze-dried to obtain the immobilized enzyme.

Embodiment 2

[0022] Put 50 mg of Uio-66(Zr) in the reaction tube, and then add 200 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at a concentration of 20 mg / mL , stirred and activated at 25°C for 1.5 h, and activated Uio-66(Zr) was initially obtained; then added 200 μL of phosphate buffer solution containing N-hydroxysuccinimide at a concentration of 12 mg / mL, and reacted at 25°C 1.5 h; finally, 3.6 mL of Pseudomonas fluorescens lipase solution containing 35 mg was added to the above mixture containing activated MOFs, crosslinked at a certain temperature for 4-20 h, centrifuged, and deionized water Washing and then freeze-drying to obtain immobilized enzyme.

Embodiment 3

[0024] Put 50 mg of Uio-66(Zr) in the reaction tube, and then add 200 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride at a concentration of 10 mg / mL , stirred and activated at 25°C for 1.5 h, and activated Uio-66(Zr) was initially obtained; then added 200 μL of 24 mg / mL phosphate buffer solution containing N-hydroxysuccinimide, and reacted at 25°C 1.5 h; finally, 3.6 mL of Pseudomonas fluorescens lipase solution containing 35 mg was added to the above mixture containing activated MOFs, crosslinked at a certain temperature for 4-20 h, centrifuged, and deionized water Washing and then freeze-drying to obtain immobilized enzyme.

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Abstract

The invention discloses an immobilization method of pseudomonas fluorescens lipase, and particularly relates to an immobilization method of lipase for chiral drug separation. The method comprises thefollowing steps of activating Uio-66 (Zr) MOF by using a phosphoric acid buffer solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, then adding N-hydroxysuccinimide, and then adding a phosphoric acid buffer solution of the pseudomonas fluorescens lipase into a mixed solution to carry out covalent cross-linking, thus obtaining a final product. According to the enzyme immobilization method, the lipase is immobilized by using a metal organic framework material, so that the activity and enantioselectivity of free enzyme can be maintained, and meanwhile, the lipase has stability and reusability. The immobilized enzyme is used for catalytic separation of a 2-(4-hydroxyphenyl) ethyl propionate enantiomer and a 4-methoxymandelic acid enantiomer, (R)-(-)-2-(4-hydroxyphenyl) propionic acid and (R)-4-methoxymandelic acid are prepared, and high catalytic activity and enantioselectivity are shown; and the method provides the wide prospect for the immobilized enzyme in industrial application.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for immobilizing lipase with a metal-organic framework material, in particular to a method for immobilizing lipase applied to chiral enantiomer resolution. Background technique [0002] Enzymes, as one of the fruits of bioengineering research, are widely used in the synthesis of chiral drugs and the production of cosmetics, detergents, food, and spices. In recent years, with the continuous expansion of the chiral drug market and the sharp increase in the demand for single chiral enantiomers, more and more separation methods have been developed. Among them, the enzyme resolution method can directly convert the chemically synthesized racemate into a single enantiomer, and has the advantages of mild conditions, high selectivity, less side reactions, less impurity components, high yield, and simple reaction operation. And it has less pollution, greatly reduces the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N11/02C12N11/14C12P7/42C12P41/00
CPCC12N9/18C12N11/02C12N11/14C12P7/42C12P41/001C12Y301/01003
Inventor 唐课文张盼良许卫凤戴桂林
Owner HUNAN INSTITUTE OF SCIENCE AND TECHNOLOGY
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