The m-hydroxybenzoate hydroxylase (MOB4-HOase) with 62 000 relative molecular mass from Comamonas testosteroni was purified to homogeneity upon SDS-PAGE by using sonic crushing, ammonium sulfate fractionation, molecular sieve chromatography, calcium phosphate gel chromatography and ion exchange chromatography. MOB4-HOase has been purified about 21 fold in specific activity with about 30% yield. This enzyme is a FAD-monooxygenase to catalyze m-hydroxybenzoate to protocatechuic acid.