BIOCHEMICAL CHARACTERIZATION OF RHODANASE FROM ASPERGILLUS WELWITSCHIAE LOT1 ASSOCIATED WITH BATTERY-EFFLUENT CONTAMINATED SOIL AND ITS BIOREMEDIATION PROPERTIES

Abstract

Rhodanese is a sulphur transferase and the principal enzyme found in single- and multicellular organisms. It is capable of mobbing up cyanide present in waste water arises from various industrial and commercial processes by catalysing the chemical from a thiosulphate to a less toxic, thiocyanate; thereby proffering remedy to environmental cyanide pollution. Hence, the objective of this study was to purify and characterize rhodanese produced from Aspergillus welwitschiae isolated from battery effluent contaminated soil and assess bioremediation properties associated with the fungus. A. welwitschiae LOT1 with the highest rhodanese-producing ability of all the fungi isolated from battery waste water polluted soil obtained from Ogunpa market, Ibadan, Nigeria was employed for enzyme production optimization using different culture parameters. The physicochemical properties of the crude rhodanese were also investigated followed by its three steps purification procedure. The physicochemical properties of purified rhodanese were also studied. Cyanide degradation efficiency of A. welwitschiae LOT1 was examined in the presence of different culture parameters while the factors yielding maximum cyanide degradation efficiency was employed for fermentation of A. wewitschiae LOT1 biomass in free and agar-agar immobilized form. The metabolites released were measured by HPLC and the structural analysis of immobilized biomass was carried out using scanning electron microscope (SEM). The results showed that rhodanase production was optimum at pH 3 and 27 oC and in the presence of fructose, casein, sugar cane baggass and 50 mM cyanide concentration. The crude enzyme revealed optima activity at pH 10 and 70 oC while maximum residual activity of 66-97% was observed at pH 2-7 and 47-65% at 25-80 oC after 1 h incubation. The enzymatic activity was inhibited by Ca2+, Cu2+, Hg2+ and EDTA but was not affected by urea at 10 and 20 mM, Km of 165 and 7.001 mM and Vmax of 0.539 and 0.146 RU/ml were obtained for KCN and Na2S2O3 respectively. An ~58 kDa purified rhodanese had specific activity of 149 RU/mg and 11.2-fold purification and 3.0% recovery. Enzymatic optimum activity was observed at pH 7 and 60 oC. It was both thermal and pH stable with maximum residual activity obtained at 30-50 oC and pH 8-10 with 72-82% and 80% respectively. All the metal ions and inhibitors investigated except Co2+ , EDTA and iodoacetamide enhanced the enzymatic activity while it showed preference for Na2S2O3, Na2SO4 and Na2S2O5 respectively. Km of 37.5 and 148 mM and Vmax 0.88 and 1.3 RU/mL. Cyanide degradation was most efficient at pH 12, 50 oC and 50 mM cyanide concentration. Sucrose and glycine of all carbon and nitrogen sources yielded maximum cyanide degradation. Other parameters such as corn stalk and locust bean as agricultural wastes, 1 mL inoculum size and 40 mM cyanide + 40 mM glucose also enhanced cyanide degradation. Whereas, there was no significant difference in the time of incubation. Under optimum conditions, A. welwitschiae LOT1 exhibited almost 100% cyanide removal of 50 mM cyanide with the release of CO2, NH3 and formamide as metabolites as revealed by HPLC coupled with scanning electron microscope (SEM) examination which also established efficient removal of cyanide. Findings revealed that cyanide biodegradation efficiency of A. welwitschiae LOT1 isolated from battery waste contaminated soil may be due to inherent cyanide-detoxifying rhodanese and established its application in heavily polluted site.