Lipase immobilized carrier and method for immobilizing lipase

A technology of immobilized carrier and lipase, which is applied in the direction of immobilizing on/in the organic carrier, can solve the problems of harsh preparation conditions, cumbersome and complicated preparation steps, etc., and achieve the effect of abundant sources, simplified preparation process and low price

Active Publication Date: 2017-12-26
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the immobilized enzyme is covalently bound, the interaction between the enzyme and the carrier is strong, and it is relatively stable in the reaction, but its preparation steps are cumbersome and complicated, and the preparation conditions are harsh.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Take 100 grams of skin collagen fiber dispersion with a mass fraction of 10%, adjust the pH value to 3.5 with 1.0Mol / L citric acid, stir well, then add 100 grams of glutaraldehyde with a mass fraction of 10%, in a water bath at 30°C, stir Cross-linking reaction for 5 hours, then slowly adjust the pH value to 7.0-8.0 with a mass fraction of 10% sodium hydroxide solution, continue the reaction for 0.5 hour, filter, and then wash with phosphate buffer solution with a pH of 7.0 for 3-5 times. Store after use or freeze-dry. The specific surface area of ​​the obtained carrier is 20m 2 / g, dry heat denaturation temperature 84.5°C, free aldehyde content 3.8mmol / g.

[0029] 1 gram of lipase (Shenzhen Lvweikang Co., Ltd.) with an activity unit of 10000U / g and 100 grams of the lipase immobilized carrier prepared above (the ratio of lipase and carrier is 100U / g carrier) were put into 200 ml of pH 7.5 in phosphate buffered saline solution at 25°C for 2 hours. After the reaction i...

Embodiment 2

[0032] Put 10 grams of cowhide collagen sponge into 90 grams of 0.5Mol / L acetic acid solution to disperse, adjust the pH value to 2.8 with formic acid, stir well, add 120 grams of succinic dialdehyde with a mass fraction of 10%, and stir in a water bath at 45 °C Cross-linking reaction for 8 hours, then slowly adjust the pH value to 7.0-8.0 with a mass fraction of 10% sodium bicarbonate solution, continue the reaction for 1 hour, filter, and then wash with Tris-hydrochloric acid buffer solution with a pH of 7.0 for 3-5 times, Store until use or freeze-dry. The specific surface area of ​​the obtained carrier is 16.3m 2 / g, dry heat denaturation temperature 89.6°C, free aldehyde content 5.0mmol / g.

[0033] 0.3 grams of lipase (Novozymes) with an activity unit of 50000U / g and 100 grams of the lipase immobilized carrier prepared above (the ratio of lipase and carrier is 150U / g carrier), put into 200 milliliters of pH 7.1 In Tris-hydrochloric acid buffer, react at 45°C for 6 hours...

Embodiment 3

[0036] Put 25 grams of degreased and depilated cowhide pieces with a mass fraction of 40% into 75 grams of deionized water, adjust the pH value to 5.0 with 1.0Mol / L formic acid, and stir well, then add 30 grams of glutaraldehyde with a mass fraction of 10% , the mass fraction is 20 grams of 10% glyoxal, in a water bath at 20°C, stir the cross-linking reaction for 6 hours, then slowly adjust the pH value to 7.0-8.0 with a mass fraction of 10% sodium carbonate solution, continue the reaction for 1 hour, filter, Then wash with phosphate buffer solution with a pH of 7.0 for 3 to 5 times, and store for later use or freeze-drying. The specific surface area of ​​the obtained carrier is 2.0m 2 / g, dry heat denaturation temperature 100°C, free aldehyde content 1.7mmol / g.

[0037] 0.2 grams of lipase (Shenzhen Lvweikang Co., Ltd.) with an activity unit of 20000U / g and 20 grams of the lipase immobilized carrier prepared above (the ratio of lipase and carrier is 200U / g carrier), put it i...

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Abstract

The lipase immobilization carrier disclosed in the present invention uses skin collagen as a base material, and after cross-linking with a cross-linking agent under acidic conditions and moderately alkaline conditions, the unreacted cross-linking agent is removed by washing with a neutral buffer solution That is, the obtained carrier has a free aldehyde group content of 0.5-5.0 mmol / g, a dry heat denaturation temperature of 80-100° C., and a specific surface area of ​​2.0-20 m2 / g. The invention also discloses a method for immobilizing lipase by using the aforementioned lipase immobilization carrier, the activity of the immobilized lipase obtained by the method is above 70U / g, and after being stored at normal temperature for one month, the enzyme activity is above 95%. The carrier raw material provided by the invention is rich in sources, naturally renewable, low in price, large in specific surface area, stable in chemical properties, high in thermal stability, low in preparation cost, high in activity and activity retention of fixed lipase, and can be stored at room temperature. months, the enzyme activity can still maintain more than 95%.

Description

technical field [0001] The invention belongs to the technical field of preparation of an enzyme immobilized carrier and its immobilized lipase in enzyme engineering, and in particular relates to a lipase immobilized carrier and a method for immobilizing the lipase. Background technique [0002] Lipase has been widely used in food, oil, biodiesel, pharmaceuticals and other fields, but if free lipase is used, it is very difficult to separate, regenerate and recycle lipase from the product in the later stage. [0003] Immobilized enzymes use physical or chemical methods to combine enzymes with water-insoluble macromolecule carriers or embed enzymes in water-insoluble gels or microcapsules of semi-permeable membranes. After the enzyme is immobilized, the general stability will increase, and it is also easy to separate from the reaction system, which is convenient for transportation and storage, and is easy to control, and can be used repeatedly, which is conducive to automatic p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/02
Inventor 程海明宋德伟陈敏李志强
Owner SICHUAN UNIV
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