Preparation method of immobilized lipase with purified cotton towel as carrier

A technology for immobilizing lipase and lipase, which is applied in the direction of being immobilized on/in an organic carrier, can solve the problems of increasing the production cost of immobilized enzyme, long immobilization period, long cross-linking time, etc., and achieves good industrialization. Application prospects, good operating stability, and the effect of short reaction times

Inactive Publication Date: 2011-10-05
SOUTH CHINA UNIV OF TECH
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  • Abstract
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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)

Method used

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  • Preparation method of immobilized lipase with purified cotton towel as carrier
  • Preparation method of immobilized lipase with purified cotton towel as carrier
  • Preparation method of immobilized lipase with purified cotton towel as carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, GA is the single factor optimization of cross-linking agent immobilized lipase

[0056] 1 Pure cotton towel absorbs 1.5~6.0mg / ml polyethyleneimine with a pH of 4.0~10.0. After the adsorption is completed, add porcine pancreas lipase aqueous solution prepared with distilled water and shake it on a constant temperature air shaker at 0~55°C. After the adsorption is completed, remove the supernatant, and then use 0.05-0.30% cross-linking agent GA with a pH of 4.0-10.0 to cross-link the towel adsorbed with enzyme and polyethyleneimine. The cross-linking time is 5-200min. After completion, take out the towel, wash the towel with pH 4.5 acetic acid buffer until no protein is eluted, dry at room temperature to obtain immobilized lipase, collect the supernatant, cross-linked solution and eluate, and measure the protein content. Then the protein load of immobilized lipase was calculated, and the best single factor condition of immobilized porcine pancreatic lipase w...

Embodiment 2

[0057] Embodiment two, the response surface experiment of immobilized porcine pancreatic lipase:

[0058]Based on the single factor experiment, the concentration of polyethyleneimine (2.0~4.0mg / ml), the concentration of glutaraldehyde (0.1%~0.3%), and the crosslinking time (10~110min) were the influencing factors, and stearic acid The specific enzyme activity (U / g towel) of transesterification between vinyl ester and pentanol was taken as the response value, and a three-factor three-level response surface experiment was designed using Design Expert 8.0.4.0 software to obtain the best condition for lipase immobilization: PEI concentration 3.37mg / ml, GA concentration 0.26%, cross-linking time 50.45min, the theoretical value of the specific enzyme activity of porcine pancreatic lipase catalyzing the transesterification reaction of vinyl stearate and amyl alcohol is 5.03U / g towel, and the actual verification value is 4.99 U / g towels.

Embodiment 3

[0059] Embodiment three, using THP as cross-linking agent immobilized lipase:

[0060] 0.2g / piece of towel, add 0.1~0.9ml 3.37mg / ml PEI solution with pH 7.0, after the adsorption is completed, add the lipase solution for esterification prepared with distilled water, and add the lipase solution at 0~40℃ with the number of revolutions of the shaker Shake at 260rpm, the solution becomes clear, pour out the supernatant, put the PEI and enzyme-adsorbed towel into 2ml of 0.05%~0.50% THP solution, crosslink at 0~40℃ for 5~200min, crosslink After completion, take out the towel, wash the towel with acetate buffer solution of pH 5.0 until no protein is washed down, and dry at room temperature to obtain immobilized lipase, which is evaluated by the enzyme activity of catalyzing the transesterification of vinyl n-butyrate and amyl alcohol index. When the concentration of THP was 0.05%, 0.10%, and 0.50%, the ester exchange enzyme activity of lipase for immobilized esterification was 16.20...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08
Inventor 魏东梁欣欣
Owner SOUTH CHINA UNIV OF TECH
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