Preparation method and application of immobilized aspergillus terreus lipase

A technology of lipase and Aspergillus terreus, which is applied in the field of preparation of immobilized Aspergillus terreus lipase, can solve the problems of poor stability, non-recyclability and low yield of Aspergillus terreus lipase, achieve good stereoselectivity and increase yield , good catalytic efficiency

Active Publication Date: 2014-03-26
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem to be solved in the present invention is that free Aspergillus terreus lipase has poor stability and cannot be recycled, and the existing ketoprofen vinyl ester hydrolysis method yield is low

Method used

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  • Preparation method and application of immobilized aspergillus terreus lipase
  • Preparation method and application of immobilized aspergillus terreus lipase
  • Preparation method and application of immobilized aspergillus terreus lipase

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preparation example Construction

[0034] The preparation method of immobilized Aspergillus terreus lipase comprises the following steps: reacting glycine protected by tert-butoxycarbonyl with mPEG2000 to obtain mPEG2000 modified by Boc-protected glycine; removing the Boc protecting group to obtain mPEG2000 modified by terminal amino group; Amino-modified mPEG2000 was reacted with sodium alginate to obtain Alg-g-mPEG; after Aspergillus terreus lipase was mixed with Alg-g-mPEG, it was reacted with α-CD to obtain Alg-g-PEG / α-CD.

[0035] As a preferred version of the present invention, the specific operation steps of the preparation method of the above-mentioned immobilized Aspergillus terreus lipase include:

[0036] a. Dissolve mPEG2000 in dry CH 2 Cl 2 Add Boc-protected glycine and DMAP, cool at -1-2°C for 10-15 minutes, then add DCC, react at -1-2°C for 18-24 hours, and the filtered solid is Boc-protected glycine-modified mPEG2000; The molar ratio of mPEG2000, Boc-protected glycine, DMAP, and DCC is 1:1.4~3...

Embodiment 1

[0075] The preparation of embodiment 1 immobilized Aspergillus terreus lipase

[0076] Add 100 μL of Aspergillus terreus lipase enzyme solution to 2 mL of Alg-g-mPEG solution (pH=7.2, Tris-HCl) with a concentration of 1% (w / v), mix well, and slowly add dropwise to 4 mL with a concentration of 6% α-CD solution, after the dropwise addition, stirred at 4°C for 12 hours, the solution became turbid and a large amount of white precipitate was formed, the mixture was centrifuged, washed three times with Tris-HCl buffer solution with pH=7.2, and the precipitate was frozen Dry to obtain immobilized Aspergillus terreus lipase.

[0077] Add 1mL of tributyrin and 3mL of 0.1M sodium phosphate buffer solution with pH=7.0 into the flask, preheat in a water bath at 30°C for 10min, add 50mg of immobilized Aspergillus terreus lipase, react for 15min, then add 8mL of toluene, vortex Mix for 2 minutes, take the supernatant and centrifuge (2000rpm, 5min), take 4mL of the supernatant into a centri...

Embodiment 2

[0078] The preparation of embodiment 2 immobilized Aspergillus terreus lipase

[0079] Add 180 μL of Aspergillus terreus lipase enzyme solution to 2 mL of Alg-g-mPEG solution (pH=7.4, Tris-HCl) with a concentration of 2% (w / v) and mix evenly, and slowly add it dropwise to 4 mL with a concentration of 9% α-CD solution, after the dropwise addition, stirred at 4°C for 24 hours, the solution became turbid and a large amount of white precipitate was formed, the mixture was centrifuged, washed three times with Tris-HCl buffer solution with pH=7.4, and the precipitate was frozen Dry to obtain immobilized Aspergillus terreus lipase.

[0080] Add 1mL of tributyrin and 3mL of 0.1M sodium phosphate buffer solution with pH=7.0 into the flask, preheat in a water bath at 30°C for 10min, add 50mg of immobilized Aspergillus terreus lipase, react for 15min, then add 8mL of toluene, vortex Mix for 2 minutes, take the supernatant and centrifuge (2000rpm, 5min), take 4mL of the supernatant into ...

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Abstract

The invention belongs to the field of chemical compounding, and particularly relates to a preparation method and application of immobilized aspergillus terreus lipase. The invention aims to solve the technical problems that free aspergillus terreus lipase is low in stability and cannot be recycled and the current ketoprofen vinyl ester hydrolysis method is low in productivity. In order to solve the technical problems, the preparation method for the immobilized aspergillus terreus lipase comprises the following steps of uniformly mixing aspergillus terreus lipase with alginate grafted methoxypolyethylene glycols (Alg-g-mPEG), and reacting with alpha-cyclodextrin (alpha-CD) to obtain the immobilized aspergillus terreus lipase. The invention also provides a method for catalyzing the hydrolysis of racemic ketoprofen vinyl ester by the immobilized aspergillus terreus lipase; the method has better stereoselectivity, the productivity is greatly improved, and the immobilized aspergillus terreus lipase can be recycled.

Description

technical field [0001] The invention belongs to the field of chemical synthesis, and in particular relates to a preparation method and application of immobilized Aspergillus terreus lipase. Background technique [0002] Free enzymes have poor stability and cannot be recycled in practical industrial applications. Enzyme immobilization technology enables enzymes to overcome these shortcomings. At present, enzyme immobilization techniques are mainly divided into four categories: adsorption method, embedding method, cross-linking method and covalent binding method. The adsorption method is to immobilize the enzyme on the carrier through ion interaction or physical adsorption. The operation is simple, the conditions are mild, and the enzyme activity is well maintained. However, due to the weak physical adsorption force, the immobilized enzyme is easy to lose, and industrial applications are limited to a certain extent. limit. The cross-linking method uses bifunctional or multif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12N11/08C12P41/00C12P7/62C12P7/40C12R1/66
CPCY02P20/50
Inventor 余孝其胡成莉王娜张晟
Owner SICHUAN UNIV
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