Functional ionic liquid modified lipase and modification method thereof

A technology of ionic liquid and lipase, which is applied in the direction of hydrolytic enzymes, etc., can solve the problems of long cycle and complicated operation, and achieve the effect of short cycle, simple operation and reduced loss

Inactive Publication Date: 2012-07-11
青州市瑞鑫再生资源科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole process includes: carrier preparation, ionic liquid synthesis, ionic liquid to modify the carrier, and then immobilize the enzyme. The reaction cycle is long and the operation is complicated. The combination of ionic liquid and enzyme is through electrostatic force and hydrogen bond.

Method used

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  • Functional ionic liquid modified lipase and modification method thereof
  • Functional ionic liquid modified lipase and modification method thereof
  • Functional ionic liquid modified lipase and modification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]1) Synthesis of 1-methyl-3-carboxyethylimidazolium chloride salt: According to Jean Pierre Bazureau et al. (Rate accelerations of 1,3-dipolar cyclodition reactions in ionic liquids. Tetrahedron Letters, 2000, 41: 7351-7355. ) to prepare 1-methyl-3-carboxyethylimidazolium chloride salt with a yield of 78.3% and a purity of 98.8%.

[0034] 2) Activation of functionalized ionic liquid: 0.17g of 1-methyl-3-carboxyethylimidazolium chloride salt, 0.192g of EDC·HCL and 0.115g of NHS were dissolved in 10ml of MES, and reacted at 30°C for 1h.

[0035] 3) Enzyme modification: The activated functionalized ionic liquid and the purified porcine pancreatic 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 modification was removed by ultrafiltration and centrifugation agent, concentrated enzyme.

[0036] 4) Determination of enzyme activity:

[0037] Lipase hydrolysis of triacetin em...

Embodiment 2

[0049] 1) Synthesis of 1-ethyl-3-carboxyethyl imidazolium chloride salt: according to Jean Pierre Bazureau et al. (Rate accelerations of 1,3-dipolar cycloloaddition reactions in ionic liquids. Tetrahedron Letters, 2000, 41: 7351-7355 .) to prepare 1-ethyl-3-carboxyethylimidazolium chloride salt with a yield of 71.6% and a purity of 97.6%.

[0050] 2) Activation of the functionalized ionic liquid: 0.18g of 1-ethyl-3-carboxyethylimidazolium chloride salt, 0.288g of EDC·HCL and 0.115g of NHS were dissolved in 10ml of MES, and reacted at room temperature for 1.5h.

[0051] 3) Enzyme modification: The activated functionalized ionic liquid and the purified Rhizopus lipase were mixed and reacted at a molar ratio of 300:1, the reaction temperature was controlled at 0-4°C, the reaction time was 6h, and excess modification was removed by ultrafiltration and centrifugation agent, concentrated enzyme.

[0052] 4) Same operating conditions as in Example 1 for preparing the modified enzyme...

Embodiment 3

[0054] 1) Synthesis of chlorinated 1-methyl-3-(3-aminopropyl) imidazole: according to the description of Wu Youting et al. 1-Methyl-3-(3-aminopropyl)imidazole chloride was obtained with a yield of 82.7% and a purity of 98.1%.

[0055] 2) Enzyme modification: Dissolve 0.18g of 1-methyl-3-(3-aminopropyl)imidazole chloride, 1.44mg of EDC·HCL, 0.58mg of NHS and 0.165g of Pseudomonas cepacia lipase in 10ml of MES, and react The temperature is controlled at 0-4°C, the reaction time is 4 hours, excess modifiers are removed by ultrafiltration and centrifugation, and the enzyme is concentrated.

[0056] 3) The operating conditions for preparing the modified enzyme in Example 1 are the same, the apparent activity of the functionalized ionic liquid modified Pseudomonas cepacia lipase is 28501U / g lipase, while the free Pseudomonas cepacia lipase The enzyme activity is 2240lU / g lipase.

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Abstract

The invention discloses functional ionic liquid modified lipase and a modification method thereof. The method comprises the following steps of: dissolving functional ionic liquid containing carboxyl, NHS and EDC.HCL in a molar ratio of 1:1:(1-3) in methyl ester sulfonate (MES), reacting at room temperature for 1 to 2 hours for activation, performing hybrid reaction on the activated functional ionic liquid and the lipase in the molar ratio of (200-1,000):1 at the temperature of between 0 and 4 DEG C for 2 to 8 hours; or dissolving the functional ionic liquid containing amino, NHS, EDC.HCL and the lipase in a molar ratio of (100-300):1:(1-3):1 in the MES, and performing hybrid reaction at the temperature of between 0 and 4 DEG C for 3 to 10 hours to obtain the functional ionic liquid modified lipase. The functional ionic liquid with the amino or the carboxyl is introduced into the surface of lipase, and is subjected to covalent binding with the amino acid residues of the lipase molecules, so the space structure of the active center of enzyme is influenced, and the catalytic performance of the lipase is enhanced.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and relates to a functionalized ionic liquid-modified lipase and a modification method thereof. Background technique [0002] Enzymes are special catalysts that can catalyze a variety of reactions under mild conditions. The biotechnology industry centered on enzymes is one of the most concerned industries in the world today (Selection and evolution of enzymes from partially randomized non-catalytic scaffold . Nature, 2007, 448, 828-831.). In the next five years, the global industrial enzyme market will grow rapidly at an average annual rate of 6.3%. By 2013, the market share of only enzyme products will reach 7 billion US dollars, and the market potential is huge. Lipase (EC3.1.1.3) is a commonly used and important enzyme in industrial production applications, which can catalyze organic synthesis reactions such as ester hydrolysis or alcoholysis, ester synthesis, transesterification and ste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/20
Inventor 胡燚黄和贾儒
Owner 青州市瑞鑫再生资源科技有限公司
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