ISOLATION AND CHARACTERIZATION OF RHODANESE PRODUCED BY KLEBSIELLA EDWARDSII FROM CYANIDE

Water bodies used for cassava processing contains cyanide which is toxic to several forms of life. The use of enzyme for the remediation of polluted environment presents advantages over traditional technologies and also over microbial remediation. Extracellular rhodanese of strain of Klebsiella edwardsii isolated from cyanide polluted stream which is used for cassava processing was studied. Biochemical characteristics of the purified enzyme, including pH, temperature profiles, kinetic parameters, substrate specificity and effect of metallic salts were also determined. Production of rhodanese correlated with the rate of bacterial growth at the death phase and the optimum incubation time for maximum enzyme production was 48 h. The enzyme was purified by 85 % ammonium sulphate precipitation and DEAE-Cellulose ion-exchange chromatography. The pure enzyme had a specific activity of 0.0473 mg^-1 with a purification fold of 4.56 and a percentage yield of 30.30 %. The Michaelis-Menten constants (Km) values of rhodanese from Klebsiella edwardsii for potassium cyanide and sodium thiosulphate were 50 mM and 200 mM respectively while their maximum reaction velocities (Vmax) were 0.56 RU/ml/min and 1 RU/ml/min respectively. The substrate specificity study showed the percentage utilization of the various substrates to be: 2-mercaptoethanol (33.7 %), ammonium persulphate (122.9 %), ammonium sulphate (178.3 %), sodium metabisulphite (67.5 %) and sodium sulphite (78.3 %). The enzyme demonstrated a broad pH range but the optimum pH was at 6.0 while the optimum temperature was 60 °C. The inhibition study on the enzyme by salts (HgCl₂, MnCl₂, NiCl₂ and ZnCl₂ and KCl) at concentrations 1 mM, 10 mM, 50 mM and 100 mM showed that the enzyme was inhibited by all the salts except for HgCl₂ at concentrations 50 mM and 100 mM. The results showed that the presence of rhodanese in the Klebsiella edwardsii suggests that the enzyme may possess functional cyanide detoxification mechanism necessary for the survival of the organism in the environment.