Purification and characterization of twopolyurethanase enzymes from Pseudomonas chlororaphis

Two polyester polyurethane (PU)-degrading enzymes from Pseudomonaschlororaphis, a bacterium that utilizes polyester PU as the sole carbon and energy source,were purified to electrophoretic homogeneity as indicated by sodium dodecyl-polyacrylamide gelelectrophoresis (SDS–PAGE). Both enzymes are extracellular, soluble proteins with molecularweight of 63,000 Da and 31,000 Da. The 63,000 Da protein exhibits both esterase and proteaseactivities toward r-nitrophenylacetate and hide powder azure respectively. The enzyme has anoptimum pH of 8.5 for esterase activity and an optimum pH of 7.0 for protease activity. The31,000 Da protein exhibits esterase activity toward r-nitrophenylacetate, butyrate and propionate,and has an optimum pH of 8.5. In addition, the enzyme activities of both proteins are heat stableafter 10 min at 100°C and are inhibited 50% by the addition of 1 mMphenylmethylsulfonylfluoride indicating both are serine-hydrolases. © 1999 Elsevier Science Ltd.All rights reserved.