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Immobilization method of D-pantoic acid lactone hydrolase

A pantolactone and hydrolase technology is applied in the field of D-pantolactone hydrolase immobilization to achieve the effects of enhancing enzyme activity, having broad application prospects and reducing reaction costs.

Inactive Publication Date: 2011-06-15
河北宝利药业有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for immobilizing D-pantolactone hydrolase, which effectively solves the problems of D-pantolactone in the industrial production process. Problems, improve the production efficiency of D-pantolactone, reduce the production cost of D-pantoolactone

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A method for immobilizing D-pantolactone hydrolase, the steps are:

[0018] (1) Take 50Kg of fermented mycelium, wash it with tap water three times in a three-legged centrifuge to remove impurities such as pigments in the culture medium; add pH 7.0, 0.02 M phosphate buffer solution, use a colloid mill to treat mycelium, and mix thoroughly; use a high-pressure homogenizer to homogenize the mycelium slurry at a pressure of 0.5 MPa and a temperature of 0°C; Centrifuge for 10 minutes at a rotational speed of 4000r / min, and take the supernatant, which is the D-pantolactone hydrolase solution, and measure the enzyme activity and volume.

[0019] (2) Get 15Kg macroporous ion exchange resin D201GF, first wash 3 times with deionized water, then soak with 4%NaOH for 4h and then wash with deionized water to neutrality, then soak with 4%HCl for 4h and then wash with deionized water until Neutral, and finally soaked with more than 2 times the volume of deionized water for later use...

Embodiment 2

[0022] A method for immobilizing D-pantolactone hydrolase, the steps are:

[0023] (1) Get 75Kg of fermented mycelium, in a three-legged centrifuge, rinse three times with tap water to remove impurities such as pigments in the medium; M phosphate buffer solution, use colloid mill to treat mycelium, and mix thoroughly; use a high-pressure homogenizer to homogenize the mycelium slurry at a pressure of 1.0 MPa and a temperature of 3°C; Centrifuge at 6000r / min for 20 minutes, and take the supernatant, which is the D-pantolactone hydrolase solution, and measure the enzyme activity and volume.

[0024] (2) Get 20Kg macroporous ion exchange resin D201GF, first wash 3 times with deionized water, then soak with 4%NaOH for 4h and then wash with deionized water to neutrality, then soak with 4%HCl for 4h and then wash with deionized water until Neutral, and finally soaked with more than 2 times the volume of deionized water for later use. Take the processed resin and resuspend it with p...

Embodiment 3

[0027] A method for immobilizing D-pantolactone hydrolase, the steps are:

[0028] (1) Take 100Kg of fermented mycelium, and wash it with tap water three times in a three-legged centrifuge to remove impurities such as pigments in the medium; M phosphate buffer solution, use a colloid mill to treat mycelium, and mix well; use a high-pressure homogenizer to homogenize the mycelium slurry at a pressure of 1.5MPa and a temperature of 5°C; Under the rotating speed of 8000r / min, centrifuge for 30 minutes, take the supernatant, the supernatant is the D-pantolactone hydrolase solution, and measure the enzyme activity and volume.

[0029] (2) Get 25Kg macroporous ion exchange resin D201GF, first wash 3 times with deionized water, then soak with 4%NaOH for 4h and then wash with deionized water to neutrality, then soak with 4%HCl for 4h and then wash with deionized water until Neutral, and finally soaked with more than 2 times the volume of deionized water for later use. Take the proce...

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Abstract

The invention relates to an immobilization method of D-pantoic acid lactone hydrolase, comprising the steps: (1) mycelium is pretreated to obtain the target enzyme liquid; (2) macroporous ion exchange resin is pretreated and added with glutaric dialdehyde solution for cross linking; (3) after that, the obtained macroporous ion exchange resin is suspended the D-pantoic acid lactone hydrolase solution and washed by de-ionized water to remove resolvase, so that the immobilized D-pantoic acid lactone hydrolase is obtained. The invention takes the macroporous ion exchange resin D201GF as a carrierand immobilizes the D-pantoic acid lactone hydrolase by two-step reaction of glutaric dialdehyde, thus overcoming the defect of the traditional direct reaction of mycelium. The immobilized enzyme granules have uniform shapes, good mechanical strength and large superficial area, so as to greatly improve the load capacity of enzyme and enhance enzyme activity; the recovery rate of enzyme activity reaches 85% in average, and the recycling times of the immobilized enzyme reaches above 50, so that the reaction cost of the resolvase is reduced. Furthermore, the method has simple technique and little investment for production equipment.

Description

technical field [0001] The invention belongs to the field of enzyme immobilization, in particular to a method for immobilizing D-pantolactone hydrolase. Background technique [0002] Pantothenic acid is a B vitamin and a component of coenzyme A. Its physiological functions include: participating in the degradation of fatty acids in the body, fatty acid synthesis, citric acid cycle, acetylation of choline, synthesis of antibodies, etc. Therefore, D-pantothenic acid is widely used in medicine, food, Feed industry, its active ingredient is D-configuration D-pantothenic acid (V B3 ). However, due to the instability of pantothenic acid, its commercial forms are mainly calcium D-pantothenate, sodium D-pantothenate, potassium, etc. The main technology for producing D-pantothenic acid is the chiral resolution technology of the intermediate DL-pantoate lactone, and the synthetic route is to prepare the intermediate (D-pantoate or D-pantoate D-pantothenic acid lactone), and then D-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/06
Inventor 宁新利别松涛李丽凤杜连祥
Owner 河北宝利药业有限公司
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