Study On The Gluconic Acid Preparation By Co-Immobilization Of Glucose Oxidase And Catalase

Glucose oxidase (GOD) is able to oxidize glucose to generate gluconic acid and H2O2 specifically. Catalase (CAT) with H2O2 as the sole substrate hydrolysis is to produce H2O and O2, and the coupling reaction of GOD-CAT immobilization has been the focus of research. Gluconic acid is a organic acid, which finds applications in the food, medicine and chemical industry. There are different approaches available for the production of gluconic acid, namely, fermentation, chemical method, electrooxidation and enzyme catalysis. Compared with enzyme catalysis, the other three methods have some de fects, like low transformation rates, high cost and byproducts. The enzy me catalysis has the merits of mild reaction condition, high product purit y and short cycle. However, the application of immobilized double enzymes in gluconic acid preparation is still relatively small and immobilized double enzymes for the reuse of enzyme, high efficiency, safety and environmental friendly advantages application of enzyme catalysis in modern organic acid production has important value of research.This paper reviews the studies of co-immobilizationof glucose oxidase and catalase, and then PVA and SA composite carrier and cross-linked enzyme aggregation (CLEAs) are used to immobilize double enzyme. On the basis of this research, combining with the characteristics of two kinds of immobilized enzyme, gluconic acid is prepared by bubbling and mechanical mixing reactor respectively, providing the reference for the large scale production of glucose acid by enzyme. The main research contents are as follows:1. PVA-SA, the composite carrier, was prepared to optimize the preparation process, and the immobilized enzyme properties were studied. The preparation conditions of enzymatic activity are:add ratio CAT:GOD=10:1, enzyme dosage is 10mg/mL, the proportion of the carrier PVA:SA=9.0:1.5,the activity of glucose oxidase retained 90%. The acid resistance and thermal stability of double enzymes immobilized is greatly improved than the free enzyme. Scanning electron microscopy (SEM) observation found that immobilized enzyme particles are in uniform pore size, internal and external connected, good structure.2. Conditions of gluconic acid were explored by enzymatic preparation using the immobilized enzyme as catalyst and the characteristics of high mass transfer rate and small enzyme damage in bubble reactor. In order to reduce substrate inhibition, gluconic acid was separated by in situ ion exchange resin. The final conversion rate of glucose is 90%, and hydrochloric acid elution concentration of gluconic acid is 85.7g/L, obtaining a good application.3. Using the model of carrier free immobilization technology-cross-linked enzyme aggregates cross-linking of GOD-CAT, the preparation of compound CLEAs, optimizing preparation conditions, and the CLEAs properties were studied. The preparation conditions of enzymatic activity are:the ratio of CAT:GOD=5:1,1.5 times volume ethanol the precipitation agent, PEG20001.0%(w/v) protective agent, 2.5% (v/v) glutaraldehyde, cross-linking time of 2h, the activity of glucose oxidase retained 80%. After double enzymes cross-linked, acid resistance increased, but when the catalytic environment temperature is higher than 60℃, inactivation of CLEAs accelerated. SEM was used to observe micro morphology, and the size of CLEAs is 70μm, and in stable structures.4. Glucose is oxidized to prepare gluconic acid using GOD-CAT cross-linked enzyme aggregates as catalyst in mechanical stirring bioreactor. The catalytic conditions were optimized by orthogonal experiment and enzyme content and substrate concentration are proved to be significant factors. In amplification experiment by fermentor, gluconic acid concentration is 145.8g/L eventually, indicating that GOD-CAT cross-linked enzyme aggregates on the enhancement of substrate inhibition ability and has the broad application prospect.