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Immobilization method for thermolysin

A technology of thermolysin and amination, which is applied in the direction of immobilization on/in the organic carrier, can solve the problems of covalent immobilization difficulties, low solubility of thermolysin, etc., to shorten the immobilization time, The effect of cost reduction

Active Publication Date: 2012-05-02
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low solubility of thermolysin (1.0-1.2mg / ml) and only 9 amino groups (thermolysin is covalently linked to the carrier through amino groups), there are great difficulties in its covalent immobilization.

Method used

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  • Immobilization method for thermolysin
  • Immobilization method for thermolysin
  • Immobilization method for thermolysin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Preparation of fatty acid methyl ester sulfonate solution of thermolysin

[0038] (1) Take 2 mg of thermolysin (free enzyme, purchased from Sigma-Aldrich Shanghai Branch, the same below), disperse it with 2 ml of MES-NaOH solution with pH 7.0, 0.02M, and place it at -4°C for half an hour , to obtain 1 mg / ml fatty acid methyl ester sulfonate solution of thermolysin free enzyme.

[0039] (2) Take 2mg of thermolysin, use 2ml containing 20mM ZnCl 2 pH 7.0, 0.02M MES-NaOH solution was dispersed, and placed at -4°C for half an hour to obtain a 1 mg / ml fatty acid methyl ester sulfonate solution of thermolysin free enzyme.

[0040] (3) Take 2mg thermolysin, use 2ml containing 3M NaCl, 20mM ZnCl 2 pH 7.0, 0.02M MES-NaOH solution was dispersed, and placed at -4°C for half an hour to obtain a 1 mg / ml fatty acid methyl ester sulfonate solution of thermolysin free enzyme.

Embodiment 2

[0041] Embodiment 2: Preparation of thermolysin free enzyme into immobilized thermolysin:

[0042] A. Preparation of fatty acid methyl ester sulfonate solution of thermolysin free enzyme: take 2mg thermolysin free enzyme, use 2ml containing 3M NaCl, 20mM ZnCl 2 pH 7.0, 0.02M MES-NaOH solution was dispersed, and placed at -4°C for half an hour to obtain a 1 mg / ml fatty acid methyl ester sulfonate solution of thermolysin free enzyme.

[0043] B. Preparation of Thermolysin Free Enzyme as Immobilized Thermolysin:

[0044] (1) Carrier MCFs-NH 2 Preparation of:

[0045] ① Hydrothermal process: first weigh P123 (triblock copolymer: poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), PEO-PPO-PEO) 5.34g and batch with water Dissolve for the first time so that it can be completely transferred to the container, then add 61.34mg of ammonium fluoride and start stirring, then add 6.2ml of TMB (1.3.5-trimethylbenzene) and 27.4ml of hydrochloric acid (36.5wt%). ...

Embodiment 3

[0050] Embodiment 3: Thermolysin free enzyme is prepared into immobilized thermolysin by microwave method:

[0051] A. Preparation of fatty acid methyl ester sulfonate solution of thermolysin free enzyme: take 2mg thermolysin free enzyme, use 2ml containing 3M NaCl, 20mM ZnCl 2 PH 7.0, 0.02M MES-NaOH solution was dispersed, and placed at -4°C for half an hour to obtain a 1 mg / ml fatty acid methyl ester sulfonate solution of thermolysin free enzyme.

[0052] B. Preparation of Thermolysin Free Enzyme as Immobilized Thermolysin:

[0053] (1) Preparation of carrier:

[0054] With embodiment 2.

[0055] (2) Activate the carrier: select 3ml of p-benzoquinone solution so that the final concentration in the reaction solution is 1.5mM and 10mg of MCFs-NH 2 Shake in a constant temperature water bath shaker at 25°C at 160rpm for 2 hours, centrifuge, wash the precipitate with 20% ethanol and pure water, and redisperse the washed precipitate in 2.6ml containing 3M NaCl, 20mM ZnCl 2 PH ...

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Abstract

The invention provides an immobilization method for thermolysin. The immobilization method comprises the following steps of: (1) activating carriers: impregnating aminated mesocellular foam silicon carriers (MCFs-NH2) in 0.5 to 2.5 mM of benzoquinone solution, carrying out oscillating activation for 1 to 3 hours, centrifuging, taking precipitate out, washing the precipitate, and dispersing the washed precipitate in MES-NaOH solution again to obtain activated carrier liquid; and (2) immobilizing enzyme: adding the activated carrier liquid in to the thermolysin in an enzyme adding amount of 40 to 100mg thermolysin / g MCFs-NH2 to obtain mixed liquid, performing electromagnetic stirring reaction on the mixed liquid in ice bath for 18 to 20 hours or irradiating the mixed liquid for 2 to 4 minutes under the condition of 20 to 50 W of microwaves at the temperature of between 0 and 7 DEG C, centrifuging, taking precipitate out, and washing the precipitate by the MES-NaOH solution and thus obtaining the immobilized thermolysin. The performance of immobilized thermolysin prepared by the method is greatly improved compared with that of free enzymes, and the immobilization time is greatly shortened from 20 hours to 3 minutes and is totally shortened by 399 times, so that the cost of the enzyme immobilization is lowered.

Description

(1) Technical field [0001] The invention relates to a method for immobilizing thermolysin. (2) Background technology [0002] As a kind of biocatalyst, enzyme is widely used in food processing, medicine and fine chemical industry because of its high selectivity, mild catalytic reaction conditions and no pollution. However, natural enzymes have poor stability, are easily inactivated, cannot be reused, and are mixed into products after the reaction, making purification difficult, making it difficult to be more widely used in industry. In addition, the isolation and purification of enzymes and their one-time use also greatly increase their cost as catalysts. Under these conditions, the concept and technology of immobilized enzymes have been proposed and developed, and have become the focus of enzyme engineering research in recent years. [0003] Enzyme immobilization is a kind of technology that binds or confines enzymes in a certain area with solid materials, and can still c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08
Inventor 谢恬陈飞飞王安明张方凯杜方川
Owner HANGZHOU NORMAL UNIVERSITY
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