| Co-immobilization is a novel technology developed in recent years, which can overcome the drawbacks of single enzyme, achieve synergistic effect or continuous reaction, further reduce the production cost and improve production efficiency. A preparation method of co-immobilized glucose oxidase(GOD)-catalase(CAT) resin and its application in preparation of sodium gluconate were reseached in this paper. The main research contents include the rapid determination method of GOD activity, the preparation method of co-immobilized GOD-CAT resin, the enzymatic properties of co-immobilized enzyme, process conditions optimization of sodium gluconate production by co-immobilized enzyme.A hydrogen peroxide electrode method for determinating GOD activity level was established. This novel method was based on the working principle of the biosensor analyzer to detect the mass concentration of glucose, the GOD activity of the sample can be measured directly using SBA-40C biosensor after calibration by GOD solution of known activity unit and linearity correction. Results showed that the optimum reaction pH is 6.5, the measurement time is 20s, the operation period is less than 60s, the RSD value of 10 times was 0.63%, range 0-100U/mL of good linearity, r=0.9991.The method is simple, rapid, accurate, specific and better repeatability, which was applicable to rapid determination of GOD activity of larger number of samples.Glucose oxidase and catalase was co-immobilized on five kinds of ion exchange resins, among them D201 showed the excellent result for immobilization. Then the enzyme was immobilized by adsorption-crosslinking method, the cross-linking agent was glutaraldehyde. The optimum co-immobilized conditions were as follows: GOD/CAT=1/1(U/U), the adsorption pH, temperature and time are 7.5,30℃ and 8 h respectively. The crosslinking temperature and time were 4℃ and 8 h respectively, and the mass fraction of the crosslinking agent(glutaraldehyde) was 1%. Under these conditions, the highest activity recovery was 30.8%, better immobilized results have been achieved.The enzymatic properties of co-immobilized enzyme:the optimum temperature and pH of co-immobilized GOD-CAT resin were 45℃ and 6.0, increased by 10"C and decreased by 1.0 compared with free enzyme respectively; its heast and pH stabilities were improved to some extent than those of the free enzyme; Km=29.48 mmol/L, was 1.42 times of free enzyme, the substrate affinity was good; the co-immobilized GOD-CAT resin has good operation and storage stabilities, the remained activity were no less than 90% after repetitive operation for ten times and 93.3% after storage for 10 weeks at 4℃.In this paper, the immobilized enzymatic production of sodium gluconate is working in mild reaction conditions, which taking 5 L fermenter as reactor, using glucose and co-immobilized GOD-CAT resin as a single substrate and catalyst. The influence on production rate of sodium gluconate of five factors are researched in this paper, the optimum reaction conditions were as follows:glucose mass concentration was lOOg/L, optimum temperature and pH were 40℃ and 6.0, Stirring speed was 175 rmp, ventilation was 12 L/min, reaction time was 35 h. Under these conditions, the glucose conversion rate can reach 100%, the molar conversion rate of sodium gluconate to glucose can reach more than 98%. The co-immobilized GOD-CAT resin has good operation stability, the remained activity was 79% after repetitive operation for 8 times.The on-line detection method of glucose concentration, temperature, pH, dissolved oxygen was established by SBA-60C fermentation online automatic analysis control system. The integration analysis of biological parameters (glucose) with fermentation index (temperature, pH, dissolved oxygen) was achieved, systematic process monitoring and control methods were established for co-immobilized enzymatic production of sodium gluconate. |
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