Jorge RODRIGUE RICANO, Mario RAMIREZ LEPE

Purifcation 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, cultivatedon benzonitrile as the sole carbon source. Nit1 was purifed to homogeneity by ion exchange and gel fltration chromatography with arecovery of 7.15% and a fold of 22.5. Te molecular weight was estimated to be 47.7 kDa and the purifed enzyme was sequenced with asystem of liquid chromatography and mass spectrometry (LC-MS). The enzyme consists of 436 amino acids with a predicted molecularweight of 47.088 kDa. The sequence revealed conserved domains for a nitrilase super family such as putative active and binding sites anda 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 inthe 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/Lin 15 min, 40 min, and 45 min, respectively. Te efect 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 afected signifcantly at 0.025 mmol/L, 0.075 mmol/L, and 0.125mmol/L concentrations. However, when both ions were combined, the activity of the enzyme decreased signifcantly.

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