Characterization of rhodanese synthesized by the wild and EMS-mutated Klebsiella oxytoca JCM1665

Abstract

Cyanide poisoning remains a significant concern for both civilian and military personnel worldwide. Rhodanese, a cyanide detoxify enzyme produced by the wild (KOJCM1665) and two selected mutant strains (KOJCM1665c and KOJCM1665d) of Klebsiella oxytoca JCM 1665 was purified and characterized using a combination of standard techniques. Extracellular rhodanese yield of the wild and selected mutants were 6.2 ± 0.6, 26.7 ± 0.7 and 18.3 ± 0.3 U/mL respectively. The overall levels of recovery of rhodanese activity and fold after purification were 49 %, 66 %, and 58 % and 2.6, 2.6, and 2.3 % for the wild, KOJCM 1665c, and KOJCM 1665d, respectively. The native molecular weights of the three enzyme preparations were 35.1, 34.9, and 34.8 kDa, while the subunit molecular weight was 35 kDa for all the enzymes. The optimum activity of the enzymes was observed at 50 °C and pH 6.0. The real km of the three enzyme preparations for KCN as a substrate were 1.03 × 10 −3 , 0.95 × 10 −3 , and 0.80 × 10 −3 M, respectively, while the km for the second substrate (Na 2 S 2 O 3 ) were 0.59 × 10 −3 , 0.49 × 10 −3 , and 0.69 × 10 −3 M, respectively. The substrate specificity study showed that the enzyme preferred sodium thiosulfate as the substrate. Metal ions such as Na + and K + had significantly greater inhibitory effects on enzyme activity. This study demonstrated the potential of enhancing K. oxytoca JCM 1665 to overexpress extracellular rhodanese, and the physicochemical properties of the enzyme hold significant promise as a target for improving the efficiency of cyanide bioremediation processes. • Extracellular rhodanese production of K. oxytoca reached a yield of 26.7 ± 0.7 U/mL. • Rhodanese purification resulted in a recovery and fold of 66 % and 2.61, respectively. • The purified rhodanese has a subunit molecular weight of approximately 35 kDa. • The enzyme activity was optimum at 50 °C and pH 6.0. • The km for substrates Na 2 S 2 O 3 and KCN were 0.49 × 10 −3 and 0.95 × 10 −3 M respectively.