Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme

Gary T. Howard, William N. Norton, Timothy Burks

A soil microorganism, designated as P7, was characterized and investigated for its ability to degrade polyurethane (PU). This bacterial isolate was identified as Acinetobacter gerneri on the basis of 16 s rRNA sequencing and biochemical phenotype analysis. The ability of this organism to degrade polyurethane was characterized by the measurement of growth, SEM observation, measurement of electrophoretic mobility and the purification and characterization of a polyurethane degrading enzyme. The purified protein has a molecular weight of approximately 66 kDa as determined by SDS-PAGE. Substrate specificity was examined using p-nitrophenyl substrates with varying carbon lengths. The highest substrate specificity was observed using p-nitrophenyl-propanate with an activity of 37.58 ± 0.21 U mg(-1). Additionally, the enzyme is inhibited by phenylmethylsulfonylfluoride and by ethylenediamine-tetra acetic acid. When grown on Impranil DLN(™) YES medium, a lag phase was noted for the first 3 h which was followed by logarithmic growth for 5 h. For the linear portion of growth between 5 and 9 h, a μ value of 0.413 doublings h(-1) was calculated. After 9 h of incubation the cell number dramatically decreased resulting in a chalky precipitate. Measurements of electrophoretic mobility indicated the formation of a complex between the PU and A. gerneri P7 cells. A hybrid zeta potential had been generated between the cells and polyurethane. Further evidence for a complex was provided by SEM observation where cells appeared to cluster along the surface of polyurethane particles and along edges of polyurethane films. Occasionally, the cells established an anchor-like structure that connected the cells to polyurethane particles.