Characterization Of Glucose Oxidases From Different Sources And Their Catalytic Applications In Sodium Gluconate Production

Glucose oxidase ?EC 1.1.3.4, GOD? catalyzes the oxidation of glucose to generate gluconic acid by utilizing oxygen with simultaneous production of hydrogen peroxide. Gluconic acid is mainly used in the food industry. Currently, Aspergillus niger is one of the most widely used for sodium gluconate production. In this study, glucose oxidases from four different sources were selected as research target, including enzymatic properties, catalysis application and isolation of A. niger^s organelles. The results are as follows:1. Enzymatic properties of glucose oxidasesPurification of glucose oxidases from four different sources was carried out using ion exchange chromatography. The molecular weight of double subunit of GOD was around 130 kDa. The properties of GODs were showed as follows:?1?Four GODs had relatively stable enzymatic properties and its pH range is from 3.0 to 8.0. ?2? With respects to temperature, enzyme activities of GODs were stable within the range of -20??50?. However, enzyme activity of GODs remarkably decreased at 55? and no enzyme activity can be detected at 60 "C. ?3? Metal ions like Fe²⁺, Co²⁺, Mg²⁺, Cu²⁺ had strong inhibitory effect on these four kinds of GODs. However, EDTA was able to activate the enzyme activity of GODs. ?4?Under the condition of temperature at 30? and pH value at 7, Km and Kcat values of four kinds of GODs have been tested, respectively, with glucose at different concentrations from 20 to 100 mmol/L. Among four GODs tested in this study, the catalytic ability of GOD NV1 purchased from Novozyme was best while thermostable GOD NV2 purchased from Novazyme was poorer. Also,2D structures of four GODs were determined using circular dichroism and their 3D structures were simulated by molecular docking. At the same time, their amino acid sequences were compared based on data obtained using Maldi-TOF-TOF, indicating there were some mutations of amino acid residues.2. Catalytic applications of glucose oxidases to produce gluconateUnpurifed and purifed GOSs were applied to produce gluconate. For the original four GODs, the productivity of gluconate of unpurified GOD NV1 was highest. However, among four purified GODs, there were few differences in terms of of the yield of gluconate. Therefore, two GODs made in China could be used instead of the imported GODs in industrial production. 3. Isolation of Aspergillus niger's mitochondria using free flow electrophoresisFADH₂, which plays the key co-enzyme roles in gluconate production by A. niger, is generated in mitochondria. Then mitochondria was separated successfully from A. niger cells using free flow electrophoresis. The optimized conditions were as follows:the voltage of 300V, current of 50 mA and temperature of 4?. This deloped method had been validated to isolate mitochondria from mesenchymal stem cells. This work plays a solid foundation to explore the functional position of mitochondria to produce gluconate in A. niger cells.