Purification and characterization of an extracellular tributyrin esterase produced by a cheese isolate, Micrococcus sp. INIA 528

Abstract

Abstract Micrococcus sp . INIA 528, a microorganism isolated from raw ewe's milk Manchego cheese, produced an extracellular tributyrin esterase. The enzyme was purified to homogeneity from culture supernatant in two chromatographic steps, with a 296-fold increase of specific activity and a 79.6% recovery of the esterase activity. The homogeneous protein was characterized biochemically. Its molecular mass was estimated to be 44.6 kDa by SDS-polyacrylamide gel electrophoresis. The amino acid composition of the enzyme was determined, but its N -terminus could not be sequenced. Optimal conditions for activity of the enzyme on α -naphtyl butyrate were 30°C and a pH of 7.5. The purified enzyme was strongly inhibited by EDTA, dithiothreitol, Cu 2+ , Zn 2+ , Ni 2+ , Fe 2+ and Co 2+ , and lost 50% activity in the presence of 7.5% NaCl. It was heat-labile, retaining only 50% of its maximum activity after 5 min at 50°C. The enzyme hydrolysed α - and β -naphtyl esters of fatty acids from C4 to C18, with preference for α -naphtyl butyrate. K m values for the hydrolysis of α -naphtylbutyrate and α -naphtylcaprilate at pH 7.5 and 30°C were 80 and 60 μ m , respectively. The maximum activity on triglycerides was recorded on tributyrin, considerably higher than on tricaprylin or trilaurin. K m values for the hydrolysis of tributyrin and tricaprylin at pH 7.5 and 30°C were 5 and 0.6 m m , respectively.