Characterization of smallest active monomeric penicillin V acylase from new source: A yeast, Rhodotorula aurantiaca (NCIM 3425)

An intracellular monomeric penicillin V acylase (PVA) of 36,000 Da exhibiting p I of 4.19, purified from newly identified yeast source, Rhodotorula aurantiaca (NCIM 3425). The enzyme was purified by hydrophobic interaction chromatography. The enzyme showed optimal activity at 45 °C and retained 80% activity after incubation at 45 °C and pH 5.5 for 1 h. The enzyme showed maximum activity at pH 5.5 and was very stable between pH 5.5–6.5 with optimum stability at pH 6.0. It exhibited 50% of its original activity after 30 min incubation at 60 °C. Enzyme hydrolyzed substrates with benzyl side chain but preferred penicillin V as primary substrate. N-terminally located serine supports the fact that it belongs to Ntn (N-terminal nucleophile)-hydrolase superfamily. The initial ten amino acid residues of R. aurantiaca PVA were identical to the initial sequence of NADH dehydrogenase (EC 1.6.99.3); however the enzyme lacks dehydrogenase activity. EGTA, EDTA, hexane and ethyl acetate stabilized the activity where as small chain alcohols inhibited it. 1,4-Dioxane, THF (tetrahydrofurane), phenol and benzyl alcohol severely inhibited enzyme activity while BME and DTT increased it. Tween 80 and Tween 20 highly enhanced the activity where as SDS and Triton X-100 inhibited it.