ACTIVE RESIDUES AND IMMOBILIZATION OF CYANIDE HYDRATASE FROM CLADOSPORIUM OXYSPORUM

Cyanide hydratase (EC.4.2.1.66) was purified from Cladosporium oxysporum with specific activity of 39.6 units mg-1 protein. The enzyme exhibited appreciable stability at both 50 °C and 60 °C. Ca2+ was the best activator for the enzyme activity where Hg2+ was the most potent inhibitor among the various tested ions. On the other hand, K+ as monovalent cation did not express any remarkable effect on enzyme activity. N-ethylmaleimide (NEM), diethylpyrocarbonate (DEPC), N-bromosuccinimide (NBS) and phenylglyoxal (PGO) inhibited the enzyme activity revealing necessity of cysteinyl, histidyl, tryptophanyl and arginyl groups, respectively for enzyme catalysis. O-Phenanthroline and ?-?-dipyridyl inhibited the enzyme activity and the inhibition was concentration-dependent. Cyanide hydratase was immobilized on Ca alginate, chitosan, silica-gel and agar-agar. The chitosan was the best bead for the immobilization. The immobilized cyanide hydratase expressed higher optimal pH and higher optimal temperature compared to the free one. The immobilized enzyme expressed storage stability at 4 ?C better than that of the free one. Cyanide hydratase was capable to degrade cyanide at 5, 10 and 15 mM particularly within the first ten hours. The results suggests that cyanide hydratase from C. oxysporum can be applied in biodegradation of polluted cyanide.