Functionalized ionic liquid used for modifying lipase, preparing method and the lipase obtained by modification

An ionic liquid, lipase technology, applied in biochemical equipment and methods, enzymes, hydrolase and other directions, can solve the problems of cumbersome product processing, lack of active functional groups in molecular structure, limited modification effect, etc., to achieve good biocompatibility The effect of stability and reactivity, saving activation time and cost, and simple and flexible method

Inactive Publication Date: 2014-12-10
ZHEJIANG GONGSHANG UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0004] At present, for most biological researchers, the application of ionic liquids in the field of biocatalysis is often to use traditional ionic liquids with relatively simple structures as catalytic reaction media or a small amount of reaction accelerators (enhancers), in order to improve enzymes. Promote reaction, such as improving product yield or reaction selectivity (Biocatalysis in Ionic Liquids.Chem Rev.2007,107(6):2757-85.), but there are many limitations in this process: 1) Traditional ionic liquid is used as a reaction medium , a large amount is uneconomical, and it makes the post-processing of the product more cumbersome, and its high viscosity weakens the reaction efficiency; 2) the lack of active functional groups in the molecular structure, the improvement of the enzymatic reaction is limited; 3) as a reaction accelerator, The type is single, and the structure lacks targeted design and effective preparation methods, which is not conducive to the study of its mechanism of action on biocatalysts
Although the chemical modification of functional ionic liquids can easily and quickly improve the catalytic behavior of enzymes and change the enzymatic characteristics, this research direction is an interdisciplinary subject, and biologists who are familiar with enzymatic research may not be able to effectively use modern organic materials. Synthetic technology means to realize the structural requirements of the diversity of enzyme molecular modifiers, so the development is slow, and the synthesis and application of functional liquids provide an effective way for this direction
[0005] In 2011, Bastien Doumèche et al. found that hydroxyl-containing ionic liquids can realize the chemical modification of formate dehydrogenase under the action of activators (Ionic liquid-inspired cationscovalently bound to formate dehydrogenase improve its stability and activity in ionic liquids.ChemCatChem.2011,3 :875-82.), but this kind of conventional ionic liquid not only has limited modification effect, but also the enzyme modification reaction time is as long as 24h, which has a great influence on enzyme activity

Method used

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  • Functionalized ionic liquid used for modifying lipase, preparing method and the lipase obtained by modification
  • Functionalized ionic liquid used for modifying lipase, preparing method and the lipase obtained by modification
  • Functionalized ionic liquid used for modifying lipase, preparing method and the lipase obtained by modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1) Synthesis of 6,7-dihydro-5H-pyrrole[1,2-α]-3-carboxyethylimidazolium bromide: 0.203g6,7-dihydro-5H-pyrrole[1,2-α ] imidazole (CAS: 59646-16-1) and 2 mL of dichloromethane (DCM) were mixed evenly, then 0.374 g of methyl bromoacetate was added, and heated to reflux for 8 h. After the reaction was finished, 3M dilute hydrochloric acid was slowly added dropwise to adjust the pH to 2, the organic phase was collected, the solvent was removed under reduced pressure, and the column was separated with a yield of 91%.

[0043] 2) Activation of functionalized ionic liquids: 0.30g of 6,7-dihydro-5H-pyrrole[1,2-α]-3-carboxyethylimidazolium bromide and 0.2g of CDI were dissolved in 5mL of DMSO, at room temperature The reaction was carried out for 2 hours to obtain the activated functionalized ionic liquid, which was untreated and refrigerated at 4°C for later use.

[0044] 3) Covalent modification of enzymes: After activation, the functionalized ionic liquid was mixed with free T...

Embodiment 2

[0050] 1) Synthesis of 6,7-dihydro-5H-pyrrole[1,2-α]-3-carboxyethylimidazolium bromide: 0.203g6,7-dihydro-5H-pyrrole[1,2-α ] imidazole and 2mL DCM were mixed uniformly, then 0.374g methyl bromoacetate was added, and the reaction was heated under reflux for 8h with a yield of 91%.

[0051] 2) Activation of functionalized ionic liquids: 0.3g of 6,7-dihydro-5H-pyrrole[1,2-α]-3-carboxyethylimidazolium bromide, 0.2g of EDC·HCl and 0.12g of NHS were dissolved in 10ml of MES was reacted at 30°C for 1h.

[0052] 3) Enzyme modification: The activated functionalized ionic liquid and Thermomyces lanuginosus lipase were mixed and reacted at a molar ratio of 200:1, the reaction temperature was 0-4°C, the reaction time was 8 hours, and concentrated by ultrafiltration and centrifugation enzyme.

[0053] 4) Same as the enzyme activity assay conditions in Example 1, the transesterification activity of the covalently modified Thermomyces lanuginosus lipase (ILs-Lipozyme) measured at 30°C was ...

Embodiment 3

[0055] 1) Synthesis of 5,6,7,8-tetrahydropyridine[1,2-α]-3-carboxyethylimidazolium bromide: 0.215g of 5,6,7,8-tetrahydropyridine[1,2 -α]imidazole (CAS: 34167-66-3) was mixed with 2 mL of DCM evenly, then 0.374 g of methyl bromoacetate was added, and heated to reflux for 8 hours, with a yield of 90.3%.

[0056] 2) Activation of functionalized ionic liquid: Dissolve 0.31g of 5,6,7,8-tetrahydropyridine[1,2-α]-3-carboxyethylimidazolium bromide, 0.28g of EDC·HCl and 0.115g of NHS in In 10ml MES, react at room temperature for 1.5h.

[0057] 3) Enzyme modification: The activated functionalized ionic liquid and Candida antarctica lipase were mixed and reacted at a molar ratio of 200:1, the reaction temperature was controlled at 0-4°C, the reaction time was 4 hours, and the excess modifier was removed by ultrafiltration and centrifugation , Enzyme concentration.

[0058] 4) Same as the enzyme activity determination conditions in Example 1, the transesterification activity of the cova...

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Abstract

The invention discloses a functionalized ionic liquid used for modifying a lipase. The structure of the functionalized ionic liquid is shown as a formula (I). In the formula (I), n is 1-7, m is 1-2, and Y is a chloride ion, a bromide ion, a tetrafluoroborate ion, a hexafluorophosphoric acid radical ion or a trifluoromethylsulfonylamine ion. By subjecting the free lipase to chemical modification with the functionalized ionic liquid, stability, activity and catalytic selectivity of the free lipase can be improved. The invention also discloses a preparing method of the functionalized ionic liquid and the lipase obtained by modification. The functionalized ionic liquid with a multiple heterocycle skeleton structure is introduced onto the surface of the lipase after modification, thus influencing the configuration of the enzyme active center and improving the catalytic effects of the lipase.

Description

technical field [0001] The invention belongs to the technical field of organic synthesis and biocatalysis, and specifically relates to a class of functionalized ionic liquids with multiple heterocyclic skeleton structures, and covalently modify lipase with such functional ionic liquids to prepare lipases with high stability, catalytic activity and reaction Selective lipase. Background technique [0002] As an important class of biocatalysts, lipase can be used to catalyze various organic reactions (such as esterification, transesterification, ammonolysis and hydrolysis reactions) to prepare a wide variety of compounds (such as alcohols, esters, acids and amines) ). Chemical modification improves the catalytic properties of the enzyme itself by introducing exogenous molecular structural fragments into the enzyme protein molecule, and combined with appropriate immobilization methods, the stability and reaction selectivity of the enzyme are improved without losing the enzyme a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D487/04C07D471/04C12N9/20
CPCC07D471/04C07D487/04C12N9/20C12N9/96C12Y301/01003
Inventor 石玉刚刘玉华
Owner ZHEJIANG GONGSHANG UNIVERSITY
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