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Immobilized lipase and preparation method and application thereof

A technology for immobilizing lipase and lipase, which is applied in the direction of immobilization on or in inorganic carriers, hydrolase, chemical industry, etc., can solve problems such as loss of enzyme activity, improve catalytic efficiency, improve mass transfer rate, avoid the effect of reunion

Inactive Publication Date: 2013-08-07
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Enzyme immobilization methods include adsorption, cross-linking, embedding, and covalent bonding. The immobilization methods of cross-linking and covalent bonding may affect the active center of the enzyme, resulting in the loss of enzyme activity during the immobilization process.

Method used

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  • Immobilized lipase and preparation method and application thereof
  • Immobilized lipase and preparation method and application thereof
  • Immobilized lipase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] This experiment illustrates the preparation process of lipase crude enzyme solution.

[0057] by Burkholderia ambifaria YCJ01 (preservation number CCTCC NO: M 2011058, patent publication number is CN102329745A, patent application number is 201110211402.2) is a production strain, in a 3L fermentation medium (medium is peptone 5g / L, yeast powder 5g / L, urea 5 g / L, olive oil 5ml / L, K 2 HPO 4 2g / L, MgSO 4 ·7H 2 O 0.5g / L, Triton X-100 0.5mL / L), inoculated at 5% inoculum size, and then cultured at 150rpm and 30°C for 12h, then increased the stirring speed to 300rpm until the entire fermentation At the end of the process, an antifoaming agent was added to prevent foam generation as appropriate during the fermentation process, and cultured at 300 rpm for 48 hours to obtain lipase YCJ01 fermentation broth. The lipase fermentation broth was centrifuged, and the supernatant was concentrated by ultrafiltration on ice through a GE 10kDa ultrafiltration membrane to obtain a cr...

Embodiment 2

[0062] This experiment demonstrates that mesoporous TiO 2 Surface pretreatment process.

[0063] Weigh about 2g of mesoporous titanium dioxide powder into a 250mL round bottom flask, add about 100mL of a mixed solution of concentrated sulfuric acid / 30% hydrogen peroxide (V / V=70 / 30) to it, and magnetically Stir, heat and reflux for 1 hour, then cool down naturally, centrifuge the mixture, discard the supernatant, wash the precipitate with deionized water until neutral, and finally dry the precipitate in a vacuum oven at 120°C for 12 hours to ensure complete water removal , to get pretreated mesoporous TiO 2 .

Embodiment 3

[0065] This experiment demonstrates the modification of mesoporous TiO with 3-aminopropyltriethoxysilane 2 and the preparation of immobilized enzymes.

[0066] Weigh 500 mg of the above-mentioned pretreated mesoporous titanium dioxide into a 100 mL round bottom flask, add 100 mL of toluene, preheat at 70 °C for 10 min, then add 0.25 mmol of 3-aminopropyltriethoxysilane, in N 2 Under protection, heat to reflux for 8h. After the reaction, the supernatant was discarded by centrifugation, and the precipitate was washed with toluene, acetone, and ethanol respectively to remove the silane coupling agent that did not react successfully. Finally, dry in a vacuum oven at 70°C for 12 hours to obtain 3-aminopropyltriethoxysilane-modified mesoporous TiO 2 .

[0067] Accurately weigh the above-mentioned 3-aminopropyltriethoxysilane modified mesoporous TiO 2 100mg in a 100mL beaker, measure 5mL of the above-mentioned concentrated lipase crude enzyme solution, dilute it to 5 times with ...

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Abstract

The invention belongs to the fields of biochemical engineering and enzyme engineering, and relates to immobilized lipase and a preparation method and application thereof. The method comprises the steps of carrying out hydrophobic modification on surface of preprocessed mesoporous TiO2 by a silane coupling agent, so as to obtain a surface-modified mesoporous TiO2 carrier; taking modified mesoporous TiO2 as a carrier to carry out immobilized preparation of immobilized lipase on lipase; carrying out aromatic ester enzymatic synthesis and enzymatic enantioseparation of a drug intermediate by the immobilized lipase in an organic phase, and carrying out purification research on the lipase by the carrier. The carrier obtained by the preparation method can selectively absorb a lipase protein molecule from crude enzyme for lipase fermentation. Therefore, the method is simple in immobilization technology, mild in condition, less in enzyme activity loss in the immobilization process, and high in active recovery rate. The prepared mmobilized lipase has good stereoselectivity and catalytic activity in the organic phase, and has wide application prospect.

Description

technical field [0001] The invention belongs to the fields of biochemical engineering and enzyme engineering, and relates to an immobilized lipase and its preparation method and application, in particular to a surface-modified mesoporous TiO 2 A method for preparing immobilized lipase as a carrier, and the application of the immobilized lipase in catalyzing ester synthesis and splitting chiral compounds in organic phase. Background technique [0002] Lipase (lipase, EC 3. 1. 1. 3, triglyceride hydrolase) is an enzyme that can catalyze the hydrolysis of natural substrate triacylglycerides to generate fatty acids, glycerol and mono- or di-glycerides. Lipase can catalyze various types of reactions such as hydrolysis, esterification, and transesterification, and has high stereoselectivity, substrate specificity, and regioselectivity. As an enzyme with multiple catalytic functions, lipase is widely used in important industrial fields such as food, energy, daily chemicals, and me...

Claims

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Application Information

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IPC IPC(8): C12N11/14C12P7/62C12P41/00C12N9/20
CPCY02P20/50
Inventor 何冰芳李文豪汪斌赵洋阳吴斌
Owner NANJING UNIV OF TECH
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