Studies on novel Oxalate Oxidase produced by an endophytic bacterium Ochrobactrum intermedium CL6

Abstract

Oxalate Oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate Oxidase (OxO) finds application in clinical assay for oxalate in blood and urine, apart from several potential industrial applications. This research work is about isolation of endophytic bacteria from the tubers of the plants Colocasia esculenta, Beta vulgaris, Ipomoea batatas and peel of Musa paradisiaca fruit for screening of OxO producing strain. A total of 49 endophytes were isolated and out of which, 4 were OxO producing strains. Based upon the OxO activity produced in nutrient medium, one strain CL6 isolated from tubers of C. esculenta was selected and identified as Ochrobactrum intermedium by 16 S rDNA sequencing. The effect of media components and process variables on the growth kinetics of the O. intermedium CL6 and on the production of the enzyme OxO showed that production is inducible and requires manganese ions in the medium, and very low fill-up volume is beneficial. Characterization of the partially purified OxO revealed many intriguing characteristics. The enzyme is thermostable and remains active for 6 h in the temperature range of 4-80°C. This enzyme is the only known OxO which did not show substrate inhibition up to a substrate concentration of 50 mM. The enzymatic activity was not adversely affected by most of the metal ions and biochemical agents (K+, Na+, Zn2+, Fe3+, Mn2+, Mg2+, Glucose, Urea, and Lactate). The enzyme appears to be a metalloprotein stimulated by Ca2+ and Fe2+. It’s Km and kcat for oxalate was found to be 0.45mM and 85 s-1 respectively. Chemical modification of OxO revealed that cysteine, carboxylates, histidine and tryptophan residues are part of the active site. A two-fold increase in Oxalate Oxidase activity was observed when histidine residues were modified with 15mM diethylpyrocarbonate. Application studies on the development of a novel enzymatic treatment method to reduce total oxalate content of the extracted starch from Taro flour resulted in 97% reduction and this treatment did not alter any of the desirable physico-chemical properties of the starch.