Abstract: Background: Use of chemical modifications to identify important amino acid residues, which are crucial for the catalytic activity of oxalate oxidase produced from novel endophytic bacterium Ochrobactrum intermedium CL6, isolated from Colocasia esculenta tubers. Methodology: Oxalate oxidase produced from a newly isolated endophytic bacterium, Ochrobactrum intermedium CL6 was purified and subjected to chemical modifications using amino-acid specific reagents. The modification reactions were optimized to find the specific reagent concentration and reaction time. Results: The results revealed cysteine, carboxylates, histidine and tryptophan residues are part of the active site. Moreover, modification of cysteine and histidine residues had resulted in the enhancement of enzyme activity. A two-fold Increase in oxalate oxidase activity was observed when histidine residues were modified with 15 mM diethylpyrocarbonate for 60 min. Histidine-modified enzyme exhibited Kcat value of 140 S–1 against 91 S–1 of native enzyme. However, catalytic efficiency did not increase substantially (1.82%) due to the counteracting increased Km value. Conclusion: Kinetic studies revealed that chemical modification of histidine residues resulted in the enhancement of turnover number (Kcat) by 1.53 times compared to native enzyme. The catalytic efficiency was 2.5 times higher than the oxalate oxidase from Barley roots showing the potential of this enzyme for clinical and industrial applications. Hence, it is worthwhile improving catalytic efficiency of OxO produced from Ochrobactrum intermedium CL6 by site directed mutagenesis.