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A controllable porous carrier-free immobilized lipase and its preparation method

A carrier-free immobilization and lipase technology, applied in the direction of immobilized enzymes, biochemical equipment and methods, enzymes, etc., can solve the problems of poor controllability, low catalytic efficiency, and complicated preparation process, and achieve regular morphology, The effect of increasing the enzyme immobilization capacity and improving the catalytic efficiency

Inactive Publication Date: 2018-08-17
GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of low load capacity, complicated preparation process and high cost, low enzyme activity, low catalytic efficiency, and poor controllability and mass transfer of immobilized enzymes existing in cross-linked enzyme aggregates. To solve the problem of high resistance, it provides a preparation method of carrier-free immobilized enzyme with controllable morphology and pore size

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Dissolve the free rhizomucor miehei lipase and bovine serum albumin (BSA) protective agent in a mass ratio of 5:1 in pH=5.8, and the molar concentration is 0.1mol / L in phosphate buffered saline solution to make 10 mg / L mL of enzyme solution, take 50mL of enzyme solution, add 100mL of 0.5mol / L calcium chloride aqueous solution, mix well, quickly add an equal amount of equimolar sodium carbonate solution of 100mL, stir magnetically at 4-25°C for 1min, and statically Set for 2 hours, the free enzyme molecules are evenly dispersed in CaCO by co-precipitation 3 Inside the particulate template agent, stable spherical templated calcium carbonate particles are formed; then add 150mL of saturated ammonium sulfate solution precipitant dropwise, shake slowly at 4-25°C for 1-2min, let stand for 2h, and carry out lipase coagulation precipitation, 3000rpm Centrifuge and wash for 3 minutes to obtain coprecipitated particles;

[0025] (2) Add 1ml of 50mmol / L dithiothreitol (DTT) s...

Embodiment 2

[0031] (1) dissolving free state Candida antarctica lipase and bovine serum albumin (BSA) protective agent in a mass ratio of 6:1 in pH=7, molar concentration is 0.2mol / L in phosphate buffer solution to make 11mg / L mL enzyme solution. Take 50mL of enzyme solution, add 100mL of 0.4mol / L calcium chloride aqueous solution, mix evenly, quickly add the same amount of equimolar sodium carbonate solution 100mL, stir magnetically for 1min at 4~25℃, let it stand for 2h, pass Co-precipitation allows free enzyme molecules to be evenly dispersed in CaCO 3 Inside the particulate template agent, stable spherical templated calcium carbonate particles are formed; then 180mL of saturated ammonium sulfate solution precipitant is added dropwise, shake slowly at 4-25°C for 1-2min, and then stand for 2h to carry out lipase coagulation precipitation, 3000rpm Centrifuge and wash for 3 minutes to obtain coprecipitated particles;

[0032] (2) Add 1ml of 50mmol / L dithiothreitol (DTT) solution to the ...

Embodiment 3

[0038] (1) Dissolve free Penicillium extensa lipase and bovine serum albumin (BSA) protective agent in a mass ratio of 10:1 in pH=8, and the molar concentration is 1mol / L phosphate buffer to make 15mg / mL enzyme liquid. Take 50mL of enzyme solution, add 100mL of 0.5mol / L calcium chloride aqueous solution, mix evenly, quickly add the same amount of equimolar sodium carbonate solution of 100mL, stir magnetically for 1min at 4-25℃, let stand for 2h, pass Co-precipitation allows free enzyme molecules to be evenly dispersed in CaCO 3 Inside the particulate template agent, stable spherical templated calcium carbonate particles are formed; then 200mL of saturated ammonium sulfate solution precipitant is added dropwise, shake slowly at 4-25°C for 1-2min, and then stand for 2h to carry out lipase coagulation and precipitation, 3000rpm Centrifuge and wash for 3 minutes to obtain coprecipitated particles;

[0039] (2) Add 1.2ml of 50mmol / L dithiothreitol (DTT) solution to the co-precipi...

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Abstract

The invention discloses a controllable porous vector-free immobilized lipase and a preparation method thereof. The preparation method of the controllable porous vector-free immobilized lipase comprises the following steps: coprecipitating lipase, calcium chloride, sodium carbonate and ammonium sulfate to prepare lipase / calcium carbonate microspheres, adding dithiothreitol for carrying out cross-linking and self-assembly of lipase, and then removing calcium carbonate by using ethylene diamine tetraacetic acid to prepare the controllable porous vector-free immobilized lipase. The preparation process of the controllable porous vector-free immobilized lipase is operated at normal temperature and pressure; the operation condition is mild; the prepared controllable porous vector-free immobilized lipase is stable in structure, controllable in shape and pore size, same in components of microparticles and high in enzyme activity, is capable of effectively reducing the mass transfer limitation of substrate molecules and improving the enzyme catalysis efficiency, and is suitable for popularization and application.

Description

technical field [0001] The invention relates to the field of carrier-bound or solid-phase enzymes, in particular to a controllable porous carrier-free immobilized lipase and a preparation method thereof. technical background [0002] Lipase catalysis has attracted more and more attention because of its mild reaction conditions, strong specificity, and high efficiency. However, using free lipase directly as a catalyst has poor operational stability, easy inactivation, and difficulty in repeated use. , The problem of complex separation and purification operations. In order to solve the above problems, people immobilized lipase on a carrier to overcome the shortcomings of free lipase. Compared with free enzyme, immobilized enzyme has higher stability, is easy to separate from the reaction system, can be reused, and is conducive to automatic production. Therefore, obtaining immobilized lipase with excellent performance is of great significance to realize efficient utilization i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N11/00
CPCC12N9/20C12N11/00
Inventor 袁振宏苗长林吕鹏梅庄新姝王忠铭罗文李惠文杨玲梅刘姝娜
Owner GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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