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Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion

A technology of immobilized enzymes and geniposide, applied in the direction of immobilization on/in organic carriers, fermentation, etc., can solve the problems of entering the transformation system and final product safety risks, poor biocompatibility, etc., and achieve enhanced stability Sex and service life, increase resistance, increase mechanical strength effect

Inactive Publication Date: 2014-04-02
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The most commonly used cross-linking agent is glutaraldehyde, but it has certain biological toxicity, poor biocompatibility, and there is a safety risk of entering the transformation system and the final product, so it is gradually used by natural biological agents with mild cross-linking reaction and good biocompatibility. Crosslinker Replacement

Method used

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  • Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion
  • Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion
  • Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion

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Experimental program
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Effect test

Embodiment 1

[0037] Preparation of immobilized enzyme beads

[0038]Prepare 100mL of 2% sodium alginate solution and 1000mL of 1% CaCl 2 Solution, prepare 4 types of immobilized particles as follows.

[0039] Bead 1: the above sodium alginate solution was added dropwise to CaCl 2 solution, and gently stirred to form calcium alginate gel pellets, which were hardened for 12 hours and washed twice with Buffer B for later use.

[0040] Bead 2: Take 20mL of the above sodium alginate solution and add 50-125mg of genipin, stir and cross-link for 5-8 hours, then add CaCl drop by drop 2 The solution is formed into a ball, and the subsequent steps are the same as the ball 1.

[0041] Bead 3: Take 80mL of the above-mentioned sodium alginate solution and add 200-500mg of genipin, stir and cross-link for 2-4 hours, then add 5-8mL of the above-prepared β-glucosidase enzyme solution, stir and cross-link for 3-5 hours , followed by dropwise addition of CaCl 2 The solution is formed into a ball, and t...

Embodiment 2

[0045] Investigate the conversion effect of immobilized β-glucosidase prepared by different methods

[0046] According to the above enzyme activity assay method, the enzyme activities of the hybrid calcium alginate-immobilized enzyme (bead 3) and hybrid manganese alginate-immobilized enzyme (bead 4) prepared above were determined to be 3.1 and 5.7 U / ml respectively. glue. Take the immobilized enzyme and the free enzyme with the same enzyme activity (34U) to carry out the conversion experiment, wherein the two immobilized beads take the same volume. The transformation system is: the substrate is 1% geniposide (dissolved in Buffer B); CaCl is added according to different situations 2 or MnCl 2 solution, CaCl 2 or MnCl 2 The concentration in the system reaches 0.1%; put 40mL of conversion solution into a 150mL Erlenmeyer flask, shake in a water bath, and the temperature is 40°C. figure 1 , taking samples regularly to determine the content of geniposide and genipin, and calcu...

Embodiment 3

[0048] SEM image analysis of immobilized pellets

[0049] The SEM images of the internal microstructure of different immobilized beads are shown in image 3 shown. With the addition of the cross-linking agent genipin and β-glucosidase enzyme solution, the internal structure of the beads 1 to 4 showed a trend of becoming more and more complex, which endowed them with better and better mechanical strength, and good mechanical strength Strength helps increase resistance to physical damage like agitation. And with Mn 2+ Replacement of Ca in calcium alginate gel 2+ , can resist the cleavage of the gel by phosphate in the enzyme solution or in the conversion system, and enhance the service life of the conversion enzyme.

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Abstract

The invention discloses a preparation method of an immobilized enzyme. The preparation method comprises the following steps: performing cross-linking on polysaccharide sodium alginate with genipin; adding beta-glucosidase, and polymerizing to obtain a polymerized solution; dropwise adding the polymerized solution into a CaCl2 solution to form immobilized beads; performing metal ion substitution on the immobilized beads in a MnSO4 solution to form hybrid manganese alginate immobilized particles. The invention further discloses a geniposide conversion method which comprises the following steps: performing continuous bioconversion on the prepared hybrid manganese alginate immobilized particles in a packed bed reactor (PBR), and hydrolyzing geniposide to prepare the genipin. The preparation method has the benefits that through an immobilization method, the immobilized enzyme is better in stability and hydrolysis performance in comparison with a free enzyme, the time for achieving the same hydrolysis effect is shortened by more than half, and the hydrolysis process is more complete; the duration time is more than 500 hours and the conversion rate is kept about 90% during the continuous conversion process in the PBR.

Description

technical field [0001] The invention relates to a preparation method of immobilized enzyme and its application in the conversion of geniposide, belonging to the field of biotechnology. Background technique [0002] Since the second half of the last century, with the innovation of preparation technology, the immobilization of enzymes and microbial cells has been greatly developed, which promotes the wide application of immobilized enzymes. Compared with free enzymes, immobilized enzymes have the advantages of easy separation of enzymes and products, high stability, continuous operation, easy control of the reaction process, reusability, and significantly reduced production costs. They have been widely used in industrial fields to enhance biocatalysis. process. [0003] According to the carrier used for immobilization, the different operation methods, and the main mechanism of immobilization, the immobilization technology can be divided into embedding method, adsorption metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12P17/06
Inventor 贡国鸿郑之明刘会王丽王鹏赵根海吴跃进
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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