Preparation method and application of carrier-free combined cross-linked glucose oxidase/catalase enzyme aggregation

A technology of glucose oxidase and catalase, applied in biochemical equipment and methods, oxidoreductase, immobilized enzyme, etc., can solve the problems of high cost of immobilization of carriers, low recovery rate of enzyme activity, etc., and achieve fine particle size , the effect of small mass transfer resistance

Inactive Publication Date: 2017-07-07
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem that the present invention also needs to solve is to provide the preparation method of the cross-linked enzyme polymer of abov...

Method used

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  • Preparation method and application of carrier-free combined cross-linked glucose oxidase/catalase enzyme aggregation
  • Preparation method and application of carrier-free combined cross-linked glucose oxidase/catalase enzyme aggregation
  • Preparation method and application of carrier-free combined cross-linked glucose oxidase/catalase enzyme aggregation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation of carrier-free co-immobilized glucose oxidase / catalase Combi-CLEAs (CAT / GOx) with bovine serum albumin (BSA) as protective agent:

[0039]Mix 1mLGOx with 1mLCAT, the enzyme activity ratio is 1:3; add 1mL BSA (0.5mg / mL) solution to the above mixed enzyme solution, mix well; then add 6mL isopropanol and 1mL glutaraldehyde (25%, v / v ), isopropanol as a precipitating agent, glutaraldehyde as a crosslinking agent, 4 ℃ constant temperature incubator 100rmp precipitation crosslinking 3h, add 200μL sodium cyanoborohydride solution (50mmol / L), continue to react for 30min and then mix the solution Centrifuge at 4°C and 8000rmp for 10min, discard the supernatant, and wash the pellet 5 times with phosphate buffer to obtain Combi-CLEAs (CAT / GOx), the recovery rate of enzyme activity is 127%, as figure 1 shown.

Embodiment 2

[0041] Preparation of carrier-free co-immobilized glucose oxidase / catalase Combi-CLEAs (CAT / GOx) with polylysine (EPL, mw3000) as protective agent:

[0042] Mix 1mLGOx with 1mLCAT, the enzyme activity ratio is 1:5; add 1mL EPL (0.2mg / mL) solution to the above mixed enzyme solution, mix well; then add 6mL isopropanol as a precipitant and 1mLPEG-CHO (molecular weight 2000KDa, 0.5mg / mL), isopropanol as a precipitating agent, PEG-CHO as a crosslinking agent, 4 ℃ precipitation crosslinking for 3h, add 200μL sodium cyanoborohydride solution (50mmol / L), after 30min, centrifuge the mixed solution for 10min , the supernatant was discarded, and the precipitate was washed 5 times with phosphate buffer to obtain Combi-CLEAs (CAT / GOx), and the recovery rate of enzyme activity was 133%, as figure 1 shown. image 3 The particle size distribution diagram of the carrier-free co-immobilized glucose oxidase / catalase Combi-CLEAs (CAT / GOx) prepared by adding polylysine was detected by a particle ...

Embodiment 3

[0044] PEG-NH 2 (Molecular weight 6000) as the preparation of carrier-free co-immobilization glucose oxidase / catalase Combi-CLEAs (CAT / GOx) of protective agent:

[0045] Mix 1mLGOx with 1mLCAT, the enzyme activity ratio is 1:15; add 1mL PEG-NH to the above mixed enzyme solution 2 (0.2mg / mL) solution, mix well; then add 6mL absolute ethanol as precipitant and 1mL vanillin (1g / L), absolute ethanol as precipitant, vanillin as crosslinking agent, 4°C for precipitation and crosslinking for 3h Then add 200 μL of sodium cyanoborohydride solution (50 mmol / L), centrifuge the mixed solution for 10 min after 30 min, discard the supernatant, wash the precipitate 5 times with phosphate buffer to obtain Combi-CLEAs (CAT / GOx), and recover the enzyme activity rate of 100%, such as figure 1 shown.

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Abstract

The invention discloses a preparation method of glucose oxidase/catalase cross-linked enzyme aggregation and application thereof for producing gluconic acid or salt. Concanavalin A (ConA), polylysine (EPL), PEG-NH2, bovine serum albumin (BSA) and trehalose or PEG10000 are added into the combined cross-linked enzyme aggregation Combi-CLEAs (CAT/GOx) to be adopted as a protective agent. According to the combined cross-linked enzyme aggregation, the enzyme activity recovery of immobilized enzyme is improved (by larger than 100%), the problem that the operation stability of the combined cross-linked enzyme aggregation is poor is effectively solved, and the service life of the combined cross-linked enzyme aggregation is prolonged. In the gluconic acid or salt producing process of the carrier-free combined cross-linked glucose oxidase/catalase enzyme aggregation, the glucose conversion rate is still kept to 90% or above after 20-batch repeated usage.

Description

technical field [0001] The invention belongs to the technical field of preparation of gluconic acid or gluconate, in particular to the application of carrier-free co-immobilized glucose oxidase / catalase in catalytic oxidation of gluconic acid or salt. Background technique [0002] Gluconic acid or salt is an important class of organic chemicals, widely used in food, medicine, chemical industry and other industries. For example, calcium gluconate is widely used in clinical and medical fields; sodium gluconate can be used to clean steel billets before coating special metals, so as to enhance the bonding force between coated metals and steel surfaces; gluconic acid is pentahydroxycaproic acid, which can be used as food additives , pharmaceutical intermediates or cleaning agents, widely used. [0003] At present, the production of gluconic acid at home and abroad is mainly through the conversion of glucose as a substrate to prepare gluconic acid. The production methods include ...

Claims

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Application Information

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IPC IPC(8): C12N11/00C12P7/58
CPCC12N9/0006C12N9/0065C12N11/00C12P7/58C12Y101/03004C12Y111/01006
Inventor 应汉杰庄伟黄金莎殷航吴菁岚周精卫陈勇陈晓春朱晨杰柳东牛欢青
Owner NANJING UNIV OF TECH
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