Immobilized cyclodextrin glucoside transferase and preparation method and application thereof

A technology of glucoside and cyclodextrin, applied in the direction of immobilization on/in organic carrier, chemical industry, sustainable manufacturing/processing, etc., can solve the problem of poor thermal stability and storage stability, large loss of enzyme activity, immobilization To solve the problem of high chemical cost, achieve the effects of high thermal stability and storage stability, cost reduction, and easy extraction

Inactive Publication Date: 2011-02-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the above method has obtained immobilized cyclodextrin glucosidyl transferase, the loss of enzyme activity after immobilization is very large, less than half of the free enzyme
The immobilized cyclodextrin glucosidyl transferase developed at present mainly has the following shortcomings: (1) poor thermal stability and storage stability, not suitable for the production of AA-2G; (2) high immobilization cost, not suitable for Industrial production

Method used

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  • Immobilized cyclodextrin glucoside transferase and preparation method and application thereof
  • Immobilized cyclodextrin glucoside transferase and preparation method and application thereof
  • Immobilized cyclodextrin glucoside transferase and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1: 2-O-glucosyl ascorbic acid assay method (HPLC method)

[0032] After the transformation liquid was centrifuged at 8000 rpm for 5 min, 1 mL of the supernatant was taken, and the volume was made up to a 10 mL volumetric flask with distilled water. Then, the samples were injected after filtration with a 0.22 μm filter membrane. Substitute the peak area into the standard curve to calculate the concentration of AA-2G.

[0033] Liquid phase instrument: Agilent 1200 liquid phase instrument

[0034] Column: Agilent SB-Aq

[0035] Flow rate: 0.5mL / min

[0036] Detector: DAD

[0037] Column temperature: 25°C

[0038] Detection wavelength: 238nm

[0039] Mobile phase: 6.86g KH 2 PO 4 , 5mL of methanol, dissolved in 1000mL of ultrapure water, and adjusted to pH 2.0 with phosphoric acid.

[0040] Definition of enzyme activity: the enzyme activity is expressed by the content of glucosyl ascorbic acid (g / L) in the transformation solution, and AA-2G is used as th...

Embodiment 2

[0041] Embodiment 2: Immobilization method 1 of cyclodextrin glucosidyl transferase

[0042] Put 0.02g of molecular sieve into 10mL of enzyme solution, then put it into a shaker at 16°C, shake at 150rpm for 10h to fully absorb, then add 100μL of 25% glutaraldehyde solution to mix and crosslink for 4h, then add 0.2g of sodium alginate and mix evenly. Put it in a refrigerator at 4°C overnight for degassing, and then use a syringe to drop it into 3% CaCl2 for hardening for 4 hours. The collected capsules were rinsed with distilled water until no protein was precipitated, and the rinsed capsules were stored in glycerin in a refrigerator at 4°C for later use.

Embodiment 3

[0043] Embodiment 3: Immobilization method 2 of cyclodextrin glucosidyl transferase

[0044] Add 0.06g molecular sieves to 10mL enzyme solution, then put it into a shaker at 16°C, shake at 150rpm for 8h for full adsorption, then add 100μL of 25% glutaraldehyde solution to mix and crosslink for 4h, then add 0.2g sodium alginate and mix evenly. Put it in a refrigerator at 4°C overnight for degassing, and then use a syringe to drop it into 6% CaCl2 for hardening for 2 hours. The collected capsules were rinsed with distilled water until no protein was precipitated, and the rinsed capsules were stored in glycerin in a refrigerator at 4°C for later use.

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Abstract

The invention discloses immobilized cyclodextrin glucoside transferase and a preparation method and application thereof. The preparation method comprises the following steps of: firstly, adding a molecular sieve into enzyme liquor; secondly, adding glutaraldehyde to crosslink mixed liquor; and finally embedding with sodium alginate and calcium chloride to obtain the immobilized cyclodextrin glucoside transferase. The immobilized cyclodextrin glucoside transferase prepared by the method has higher thermal stability and storage stability, can be immobilized by crude enzyme liquor and greatly reduces immobilization cost. The immobilized enzyme can be used for producing 2-O-glucosyl ascorbic acid, the conversion rate is up to 0.22 g*L<-1>*h<-1>, products after conversion are easy to extract, and the immobilized enzyme is also easy to recycle and can be reused.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, in particular to an immobilized cyclodextrin glucosidyl transferase and its preparation method and application Background technique [0002] L-ascorbic acid (abbreviated as VC) plays an important physiological role in the body, but its reducing property is strong and extremely unstable, which limits its application. The derivatives of VC greatly improved its stability without affecting its physiological function. This patent concerns the biosynthesis of one of its derivatives, 2-O-glucosyl ascorbic acid, under the action of glycosyltransferase. [0003] 2-O-glucosyl ascorbic acid has wider application value than L-ascorbic acid, and its biotransformation synthesis method is the only production method. In 1990, the Hayashibara Institute of Biochemistry in Japan and the Department of Pharmacy of Okayama University jointly discovered 2-O-glucosyl ascorbic acid (AA-2G), and have determined a l...

Claims

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Application Information

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IPC IPC(8): C12P19/60C12N11/10C12N11/04
CPCY02P20/50
Inventor 陈坚堵国成李江华刘龙张子臣
Owner JIANGNAN UNIV
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