Cloning, expression and characterization of PURase gene from Pseudomonas sp. AKS31

Polyurethane (PUR) is a soil and aquatic contaminant throughout the world. Towards bioremediation, in a previous study, a soil bacterium, Pseudomonas sp. AKS31, capable of efficiently degrading PUR was isolated. Polyurethanase (PURase) enzyme is capable of cleaving the ester bond of PUR and is considered as a key regulator of PUR biodegradation. Hence, for a high yield, easy purification, and further characterization, the aim of this study was to clone and overexpress the PURase gene of this isolate. The current study also investigated structural aspects of this enzyme through predictive bioinformatics analyses. In this context, the PURase gene of the isolate was cloned and expressed in E. coli using pET28(a)⁺ vector. The obtained recombinant protein was found insoluble. Therefore, first, the protein was made soluble with urea and purified using nickel-NTA beads. The purified enzyme exhibited substantial activities when tested on the LA-PUR plate. Bioinformatics-based analysis of the protein revealed the presence of a lipase serine active site and indicated that this PURase belongs to the Family 1.3 lipase. Hence, the present study shows that active PURase can be produced in large quantities using a prokaryotic expression system and thus, provides an effective strategy for in-vitro PUR-degradation.