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Purification and characterization of a cyanide-degrading nitrilase from Trichoderma harzianum VSL291

An intracellular nitrilase (Nit1) with cyanide-degrading activity was isolated from Trichoderma harzianum VSL291, cultivated on benzonitrile as the sole carbon source. Nit1 was purified to homogeneity by ion exchange and gel filtration chromatography with a recovery of 7.15% and a fold of 22.5. | Turkish Journal of Biology http://journals.tubitak.gov.tr/biology/ Research Article Turk J Biol (2015) 39: 248-257 © TÜBİTAK doi:10.3906/biy-1406-51 Purification and characterization of a cyanide-degrading nitrilase from Trichoderma harzianum VSL291 Jorge RICAÑO-RODRÍGUEZ, Mario RAMÍREZ-LEPE* Unit for Research and Development in Food, Technological Institute of Veracruz, Veracruz, Mexico Received: 17.06.2014 Accepted: 02.10.2014 Published Online: 01.04.2015 Printed: 30.04.2015 Abstract: An intracellular nitrilase (Nit1) with cyanide-degrading activity was isolated from Trichoderma harzianum VSL291, cultivated on benzonitrile as the sole carbon source. Nit1 was purified to homogeneity by ion exchange and gel filtration chromatography with a recovery of 7.15% and a fold of 22.5. The molecular weight was estimated to be 47.7 kDa and the purified enzyme was sequenced with a system of liquid chromatography and mass spectrometry (LC-MS). The enzyme consists of 436 amino acids with a predicted molecular weight of 47.088 kDa. The sequence revealed conserved domains for a nitrilase super family such as putative active and binding sites and a Glu-Lys-Cys catalytic triad. Nit1 exhibited maximum activity (19.6 U mg–1) at 40 °C and a pH of 7.5. Nit1 had a strong inhibition in the presence of Al3+, Cu2+, Zn2+, and Ag+ ions and was able to degrade KCN completely at 0.02 mmol/L, 0.05 mmol/L, and 0.1 mmol/L in 15 min, 40 min, and 45 min, respectively. The effect on KCN (0.02 mmol/L) degradation was tested in the presence of Cu2+ and Ag+ ions (0.025 mmol/L to 1.0 mmol/L) and the enzymatic activity was not affected significantly at 0.025 mmol/L, 0.075 mmol/L, and 0.125 mmol/L concentrations. However, when both ions were combined, the activity of the enzyme decreased significantly. Key words: Cyanide-degrading nitrilase, bioremediation, enzyme characterization, purification, Trichoderma 1. Introduction Nitrilase enzymes (nitrilases) catalyze the hydrolysis of nitrile (R-CN) .