MICROBIAL PRODUCTION OF GLUCOSE OXIDASE AND ITS COMMERCIAL APPLICATIONS

Abstract

Glucose oxidase (EC 1.1.3.4) is an important enzyme that oxidizes glucose to gluconic acid. It is present in all aerobic organisms and has become a very useful enzyme for its wide applications especially in food industry and in clinical analysis. The most important application for GOX is the determination of glucose using biosensor technology. GOX belongs to a large group of enzymes oxido reductase and is also called as glucose aerodehydrogenase Glucose oxidase was produced from different microorganisms. Both fungi and bacteria produce glucose oxidase during fermentation. The present project was planned for the optimum production of glucose oxidase by Aspergillus niger and its utilization for estimation of glucose and for the production of calcium, gluconate, gluconic acid and its derivatives. The project was divided into two parts, in the first part production of glucose oxidase from Aspergillus niger was investigated and the second part consists of commercial applications of glucose oxidase. Here the aim was to improve GOX production using mutagenesis of A. niger, to optimize the conditions of fermentation, screen fungal strains producing highest GOX activity, and to medium composition. Mutagenesis was carried out on several strains at different time intervals. GOX enzyme purified by (NH 4 ) 2 SO 4 precipitation technique was dialysed and subjected to gel filtration chromatography. The enzyme was found to be intracellular. Five strains of A. niger isolated from grapes, bread, potato, pickle and sugar beet sources were screened for maximum GOX production. It is clear from our results that the A. niger strain isolated from potato was best for GOX production. This strain showed the maximum enzyme activity in medium containing 10% (w/v) glucose and at pH 5.5. Different conditions like the fermentation period, varying concentrations of urea, MgSO 4 .7H 2 O, CaCO 3 and KH 2 PO 4 were optimized by conducting different experiments. The maximum activity of glucose oxidase was recorded after 48 hours of continuous shaking fermentation of optimum growth medium containing 3.5% (w/v) CaCO 3 , 0.2% (w/v) Urea, 0.4% (w/v) KH 2 PO 4 and 0.01% (w/v) MgSO 4 .7H 2 O. It was observed that addition of Urea, CaCO 3 , and KH 2 PO 4 in the medium enhanced the GOX production whereas addition of MgSO 4 .7H 2 O decreased the GOX production. The GOX was found PDF created with pdfFactory Pro trial version www.pdffactory.comto be quite active upto 60 o C with optimum temperature at 30 o C. The batch fermentation volume of 50 ml at 100 rpm speed shaker was found to be the optimum for GOX production. Among mutant, it was found that mutant (9) had maximum activity and growth. The UV induced mutation gave a stable and viable culture for hyper production of GOX as the production was enhanced. Then the enzyme was purified by (NH 4 ) 2 SO 4 precipitation technique, Dialysis and Gel filtration chromatography. It was observed that enzyme activity was increased by increasing (NH 4 ) 2 SO 4 concentration. Enzyme activity also increased by Dialysis and Gel filtration chromatography from 11.90 to 37.24 μ/ml. Purification was 11.55 folds than simple precipitation at this final step. In the second part of project two commercial applications of GOX were investigated i.e. estimation of glucose by standardization of conditions using GOX and the production of calcium gluconate, gluconic acid and its derivatives using GOX. In the first application the three enzymes GOX, mutarotase (EC # 5.1.3.3) and peroxidase (EC # 1.11.1.) were produced, extracted and purified for the preparation and optimization of glucose estimation kit. The enzyme concentrations of 5 μL mutarotase, 15 μL glucose oxidase and 10 μL of peroxidase with chromagen Guaiacol added before peroxidase, proved to be best for estimations of glucose in blood samples. The sensitivity of the best kit was as low as 50 mg/dL glucose. The wavelength of 470 nm was best for the test. The results were comparable with standard kit of Medisense Abbott (UK). In the second application, calcium gluconate and gluconic acid and its derivatives were produced by glucose oxidase from Aspergillus niger. The time course during fermentation showed that the calcium gluconate production was maximum at 48 hours after conidial inoculation. The cultural conditions optimized for maximum calcium gluconate production were, glucose concentration 10% (w/v), pH 5.5, 7% (w/v) CaCO 3 , 0.2% (w/v) urea 0.15% (w/v) KH 2 PO 4 concentration at 35 o C. Different nitrogen, phosphate and metal carbonate sources were also optimized. The present study also described the production of gluconic acid and its derivatives on the laboratory scale. Gluconic acid and its metal salts such as sodium, magnesium, copper and nickel gluconates were synthesized from calcium gluconate which was produced by fermentation. The gluconic acid was released by the action of oxalic acid and sulphuric acid on calcium gluconate. Sulphuric acid gave better yields i.e. (90%) as compared to oxalic acid (80%). So the organic acid was obtained by PDF created with pdfFactory Pro trial version www.pdffactory.comH 2 SO 4 in the present work because it was cheap and readily available in local market. Metal gluconates were also produced by both the double decomposition and gluconic acid methods respectively. It is clear from the study that the gluconic acid method gave greater yields compared to the double decomposition method. This project will help in the commercial production of products using GOX in Pakistan.