Production of oxidase enzymes for clinical assyas

Abstract

The oxidase enzymes have a high potential in biotechnological applications. Screen for a good source of a particular oxidase has been actively carried out in various laboratories by using plating technique and liquid culture technique. In this investigation, nutrient selective medium technique was used to screen for microorganisms which produced choline oxidase, glucose oxidase, L-α-glycerophosphate oxidase, sarcosine oxidase and urate oxidase. Sarcosine oxidase and urate oxidase producing micro-organims were successfully screened from 50 soil samples whereas microorganism producing choline oxidase, glucose oxidase and L-α-glycerophosphate oxidae were not found. Sarcosine oxidase and urate oxidase producing microorganisms were identified as a genus Arthrobacter based on Bergeys manual and arbitarily name Arthrobacter sp S-1 and Arthrobacter sp U-1, respectively. Arthrobacter sp S-1 produced only intracellular sarcosine oxidase with specific activity about 0.06 unit/mg protein. Crude sarcosine oxidase was purified 20 folds, 72% recovery with a specific activity of 1.2 unit/mg protein. Molecular weight of this sarcosine oxidase was about 190,000 daltons estimated by gel filtration. Optimal pH and optimal temperature were about 7.7 and 45 degree C, respectively. This enzyme was stable upto 30 degree C in phosphate buffer pH 7.5 and it was stable at 4 degree C in the presence of NaN(,3). K(,m) was about 7.0 mM and it was specific to sarcosine. Effects of various activators and inhibitors were studied. Arthrobacter sp U-1 produced both intracellular and extracel-lular urate oxidase in the ratio of 20:1 and about 0.34 unit/mg protein of crude intracellular urate oxidase was obtained. This crude enzyme was purified 15 folds, 70% recovery with a specific activity of 5 unit/mg protein. The molecular weight of this urate oxidase was about 120,000 daltons estimated by gel filtration. Optimal pH was about 9.0. Optimal temperature was about 35 degree C in phosphate buffer pH 7.5 and about 50 degree C in the presence of sodium borate. This enzyme was stable upto 35 degree C in phosphate buffer pH 7.5 and 45 degree C in the presence of sodium borate. It was stable at 4 degree C in the presence of NaN(,3). K(,m) was about 7.0 x 10(,-5)M and it was specific to uric acid. Effects of various activators and inhibitors were studied. It was concluded that both enzymes from the two microorganisms are similar to the commercially available enzyme.