Preparation method of immobilized lipase with purified cotton towel as carrier

Abstract

The invention provides a preparation method of an immobilized lipase with a purified cotton towel as a carrier. The method is an immobilization technique comprising the following steps of: adsorbing a polyethyleneimine aqueous solution with the purified cotton towel, adding a lipase solution after adsorption, vibrating with a constant-temperature shaking table, thus attaching the lipase to the purified cotton towel containing polyethyleneimine, removing the supernatant, and then cross-linking the purified cotton towel with the lipase and polyethyleneimine adsorbed by using a cross-linking agent. According to the invention, the towel carrier used by the preparation method has multiple gaps and long towel fibers, thus having a larger surface area, favoring the performing of immobilized-enzyme-catalyzed reaction, having very good mechanical strength, and the method can more easily separate the enzyme from the reaction system, compared with the granular carrier; the used cross-linking agent is tris(hydroxymethyl)phosphine which has a better cross-linking effect than glutaraldehyde; the used immobilization method has the advantages of mild conditions and multi-layer immobilization; and the obtained immobilized lipase has good temperature stability and high reaction activity, can shorten the reaction time and can be repeatedly used.

Description

technical field [0001] The invention relates to the technical field of immobilized lipase, in particular to a preparation method of immobilized lipase with pure cotton towel as carrier. Background technique [0002] Lipase (Lipase, EC 3.1.1.3), also known as triacylglycerol acyl hydrolase, can catalyze the hydrolysis of natural oils to generate fatty acids, glycerol, monoglycerides or diglycerides, which is an indispensable and important hydrolysis of lipid metabolism in organisms enzyme. Lipase is a kind of biocatalyst. Its most prominent feature is that it can catalyze the reaction at the oil-water interface. Compared with chemical catalysts, it has the advantages of mild reaction conditions, high catalytic efficiency, strong specificity, and low pollution. However, in actual use, free enzymes have disadvantages such as easy inactivation, unstable catalytic reaction, difficult separation from products, or high separation costs even under optimal reaction conditions. In a...

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Problems solved by technology

Its shortcoming is that the lipase is immobilized by the adsorption method, the binding force between the enzyme and the carrier is not strong, and it is easy to fall off [Chinese patent application publication CN145667A, the publication date is 2003.11.19]

Its shortcoming is that the reaction of this method is violent, the cross-linking time is long, and it is easy to cause enzyme inactivation during the reaction (Liu Xinxi, Peng Lifeng. Immobilization of lipase by sodium periodate method on cotton fiber membrane [J]. Journal of Hebei Normal University ( Natural Science Edition) 2001,25(1):80-83.)

Its disadvantage is that the carrier treatment needs to be treated at a high temperature of 70-95 °C for 12 hours, and the energy consumption is high; ultrasonic treatment is required, and organic solvents (cyclohexane, n-heptane, n-hexane) are required for cleaning, which improves the production of immobilized enzymes Cost; the immobilization period is too long [Chinese patent application, publication number is CN101260396A, publication date 2008.09.10]

PEI is first adsorbed on the surface of the carrier, and the enzyme is electrostatically adsorbed. GA cross-links the PEI-enzyme aggregate and covers it on the surface of the support to achieve immobilization. It will also remain above 90%, and its thermal stability has also increased by at least 10-20%. However, in the crosslinking reaction, the aldehyde group reacts with the amino group to form a C=N Schiff bond, which is easy to hydrolyze, especially at higher temperatures. More seriously, the enzyme activity is greatly lost during the immobilization process; many different substances are formed due to automatic polymerization in storage, and the properties are unstable; in addition, glutaraldehyde is also a toxic substance, which is harmful to the operator (US Patent No. US7166451 , the application date is 2004.02.24)

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