Purification and characterization of poly(3-hydroxylbutyrate) depolymerase from a fungal isolate, Emericellopsis minima W2

Abstract

The fungus, Emericellopsis minima W2, capable of degrading poly(3-hydroxybutyrate) (PHB) was isolated from a waste water sample. Production of the PHB depolymerase from E. minima W2 (PhaZEmi) was significantly repressed in the presence of glucose. PhaZEmi was purified by column chromatography on Octyl-Sepharose CL-4B and Sephadex G-100. The molecular mass of the PhaZEmi, which consisted of a single polypeptide chain, was estimated to be 48.0 kDa by SDS-PAGE and its pI value was 4.4. The maximum activity of the PhaZEmi was observed at pH 9.0 and 55°C. It was significantly inactivated by 1 mM dithiothreitol, 2 mM diisopropyl fluorophosphate, 0.1 mM Tween 80, and 0.1 mM Triton X-100, but insensitive to phenylmethylsulfonyl fluoride and N-ethylmaleimide. The PhaZEmi efficiently hydrolyzed PHB and its copolyester with 30 mol% 3-hydroxyvalerate, but did not act on poly(3-hydroxyoctanoate). It also hydrolyzed p-nitrophenylacetate and p-nitrophenylbutyrate but hardly affected the longer-chain forms. The main hydrolysis product of PHB was identified as a dimer of 3-hydroxybutyrate.