Isolation and Characterization of Rhodanese from Bacillus licheniformis Obtained from Industrial Effluents of Steel Rolling Industry in Ilorin

Abstract

Pollution from industrial effluents and anthropogenic sources is becoming a very big ecological problem in Nigeria. A typical example of such pollutants is cyanide which can be degraded to a less toxic compound by some microorganism. Some facultative anaerobic bacteria such as Bacillus licheniformis utilizes cyanide as a substrate for survival in industrial contaminated and polluted ecosystem.  This study screened for bacterial from industrial effluents in Ilorin that can synthesize rhodanese, an enzyme that can degrade cyanide to less toxic substance.

Bacillus licheniformis was isolated and biochemically characterized. Rhodanese was partially purified using 85% ammonium sulphate precipitation, ion exchange chromatography on Sephadex CM-50. The molecular weight of the enzyme was determined on a gel filtration column (Sephadex G-100) and finally on Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The kinetic parameters (K_m and V_max) were determined. Statistical analysis was performed using one-way analysis of variance and results were considered significant at p<0.05.

The result of this study showed that the optimum growth temperature for B. licheniformis was 35^oC at a pH 9.0 while the highest duration time for the synthesis of rhodanese was 39 hours. Potassium cyanide (KCN) and casein were the best carbon and nitrogen sources. The B. licheniformis isolate synthesizes rhodanese that had a specific activity of 13.63 RU/mg, with a purification fold of 5.19, a percentage yield of 13.96% and an estimated native molecular weight of 34.44 kDa. The apparent   K_m for KCN and Sodium thiosulphate (Na₂S₂O₃) were determined to be 32.9 mM and 21.0 mM respectively while their V_max were 5.6 RU /ml/min and 6.4 RU /ml/min respectively.  The optimum pH and temperature of partially purified enzyme were 8.0 and 50^oC respectively. The enzyme showed high stability at 50^oC for 30 minutes. The enzyme showed specificity at 6.78 RU/ml/min and equally showed specificity for Na₂S₂O₃ while it was inhibited by other sulphur containing compounds such as 2-mercaptoethanol (2-MCPE), ammonium persulphate (NH₄)₂S₂O₈), and sodium metabisulphite (Na₂S₂O₅). The enzyme activity was not inhibited by metal ions such as (K⁺, Mg²⁺_, Ba²⁺_, Ni²⁺, Sn²⁺ and Na⁺) at 1 mM and 10 mM and this was not significant (p>0.05).

The high level of expression of rhodanese in B. licheniformis suggests that the enzyme possess functional cyanide detoxification mechanism, a bioremediation process for the survival of both plants and animals in the environment. Microbial enhancement of this kind is recommended to help in reduction of toxic materials from industrial effluents.