Growth of Bacillus subtilis on polyurethane and the purification and characterization of a polyurethanase-lipase enzyme

A soil microorganism capable of degrading polyurethane was isolated from a mesocosm study. This organism was identified as Bacillus subtilis through 16s rRNA sequencing. The ability of this organism to degrade polyurethane was characterized by the measurement of its growth kinetics and the purification and characterization of a polyurethane degrading enzyme. The growth kinetics for this microorganism was obtained on two substrates: Impranil DLNTM and tributyrin. The growth of B. subtilis on polyurethane at certain concentrations (1.5–0.18 mgml−1) and tributyrin at all concentrations followed simple Monod growth kinetics. Lineweaver–Burke analysis of the data was performed to yield a μmax value of 0.7626±0.04799 doublingsh−1 and Ks value of 0.08559±0.03291 mgml−1 for Impranil DLNTM and a μmax value of 0.46003±0.05407 doublingsh−1 and Ks value of 0.04065±0.2336 μM for tributyrin. At higher concentrations of Impranil DLNTM (9.0–3.0 mgml−1) Monod kinetics were not observed due to the binding of polyurethane to the cell wall of the B. subtilis. The purified protein as determined by SDS-PAGE has a molecular weight of approximately 28 kDa. Substrate specificity was examined using p-nitrophenyl substrates with varying carbon lengths. The highest substrate specificity was observed for p-nitrophenyl-acetate with an activity of 45 Umg−1. Additionally, the enzyme is heat labile and not inhibited by phenylmethylsulfonylfluoride (PMSF), adenylmethylsulfonylfluoride (AMSF), ethylenediamine-tetra acetic acid (EDTA), or Bromelain.