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Process for preparing chitosan microsphere immobilized lipolytic enzyme

A technology for immobilizing lipase and chitosan microspheres, applied in the direction of immobilization on/in organic carriers, hydrolytic enzymes, etc., can solve the problems of poor carrier flow rate, small surface area, low affinity of immobilized enzymes, etc. The effect of improving activity recovery rate and reducing reaction cost

Inactive Publication Date: 2011-06-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantage of using natural chitosan as a carrier to immobilize enzymes is that it is difficult to recover the immobilized enzymes due to dissolution under acidic conditions, most of which are in powder form, the carrier has poor flow rate and small surface area, which limits its application
[0005] At present, most of my country adopts a single method of immobilization, such as adsorption method or cross-linking method to immobilize lipase alone. Due to the irregular shape of the carrier, the recovery rate of immobilized enzyme activity is generally low (36%-50%) and the immobilized enzyme has a negative effect on lipase. Inadequacies such as low substrate affinity limit the application of immobilized lipase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The preparation of embodiment 1. chitosan microspheres

[0024] Weigh 1 g of chitosan and dissolve it in 2% acetic acid solution by volume fraction to prepare chitosan acidic sol with a mass fraction of 2.5%. Under stirring, slowly add the above sol into the mixed solution of 80mL liquid paraffin and 5mL Span-80, stir for 20min to disperse the sol droplets evenly, then add 10mL of 25% glutaraldehyde solution by volume fraction, stir for 10min, then use Adjust the pH to 9 with 1mol / L NaOH solution, keep it in a water bath at 70°C for 3 hours, let it stand for cooling, discard the upper oil layer, then soak it with petroleum ether, acetone, and absolute ethanol in sequence, and dry it in vacuum at 50°C to obtain Chitosan microspheres.

Embodiment 2

[0025] Example 2. Glutaraldehyde is used as a cross-linking agent to prepare immobilized lipase

[0026] Weigh 0.50g chitosan microspheres, add Na 2 HPO 4 -KH 2 PO 4 100 mL of 0.75 mg / mL Candida Rugosa lipase (Candida Rugosa) solution prepared in buffer solution to make the final concentration of chitosan microspheres 0.005 g / ml, and stirred at room temperature for 1 h. Then add 11.1 mL of 25% glutaraldehyde solution to make the final concentration of glutaraldehyde 2.5% g / ml, continue to stir for 6 hours, fully cross-link, rinse with deionized water for 3 times, filter, and dry to obtain immobilization Lipase. The activity of the immobilized enzyme reaches 473U / g carrier, the specific activity reaches 22.1U / mg protein, and the activity recovery rate reaches 38.1%.

Embodiment 3

[0027] Example 3. Activation with ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride to prepare immobilized lipase again

[0028]The chitosan microsphere lipase that takes the glutaraldehyde immobilization in 0.50g step 2 is immersed in the lipase liquid (pH=7) of 100mL volume 0.75mg / mL, makes the chitosan microsphere immobilized by glutaraldehyde The lipase concentration is 0.005g / ml, and stirred for 1h. Then add 25mL of ethyl[3-(dimethylamino)propyl]carbodiimide hydrochloride solution (0.2%) to make ethyl[3-(dimethylamino)propyl]carbodiimide The acid salt concentration is 0.04% g / ml, continue to shake for 6 hours, make it fully fixed, rinse repeatedly with deionized water 3 times, filter, and dry to obtain chitosan microspheres immobilized lipase, and the activity of the immobilized enzyme reaches 813U / g carrier, the specific activity reaches 35.3U / mg protein, and the activity recovery rate reaches 60.2%.

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Abstract

A preparation technique of chitosan microspheres immobilization lipase comprises the steps that: (1) lipase from candida SP is dissolved in phosphate buffer solution by taking chitosan microspheres as an immobilization carrier and adopting glutaraldehyde cross-linking, lipase A is obtained. (2) candida SP lipase solution of 0.70-0.80mg / mL prepared by phosphate buffer solution b with a pH value of6.5-7.5 is added with immobilization lipase A (final concentration is 0.005-0.0055g / ml) and after being stirred, ethyl (3-dimethylamino propyl) carbodiimide hydrochloride solution is added, the mixture is then stirred to be fully immobilized, chitosan microspheres immobilization lipase B is obtained after the treatment of the mixture. The chitosan microspheres immobilization lipase prepared by the preparation technology of the invention improves apoenzyme combination rate and activity recovery, has powerful immobilization lipase activity and specific activity and the immobilization lipase canbe recycled for six times.

Description

(1) Technical field [0001] The invention belongs to the technical field of biochemical industry and relates to a preparation process of chitosan microsphere immobilized lipase. (2) Background technology [0002] Chitosan is an N-deacetylated glucose polymer with abundant sources and cheap price, and is the most important derivative of chitin. Chitosan has non-toxic, good gelling properties, biocompatibility, protein affinity and metal ion chelating properties, and is a good immobilized enzyme carrier. There are many kinds of immobilized enzymes with chitosan as the carrier, such as pepsin, glucose oxidase, L-asparaginase, β-mannanase and so on. The main disadvantage of using natural chitosan as a carrier to immobilize enzymes is that it is difficult to recover the immobilized enzymes due to dissolution under acidic conditions, most of which are in powder form, the carrier has poor flow rate and small surface area, which limits its application. Therefore, the current resear...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/06C12N9/20
Inventor 孙培龙邵平孟祥河
Owner ZHEJIANG UNIV OF TECH
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