Cloning, Overexpression and Application of Lipase from Thermotolerant Bacillus subtilis TTP-06 in the Degradation of Polyhydroxyalkanoate

Abstract

Polyhydroxyalkanoates (PHAs) constitute a principal group of bio-degradable polymers that are produced by certain microbes under limited supply of nutrients. PHA is a linear polyester that comprises of 3-hydroxy fatty acid monomers. Triacylglycerol acylhydrolases are known to catalyze the hydrolysis of ester linkages and in turn they are beneficial in the degradation of PHA. In present study, lipase-catalyzed degradation of PHA synthesized by Priestia megatarium POD1 was monitored. A gene from thermotolerant Bacillus subtilis TTP-06 that was capable of expressing lipase enzyme was amplified by PCR, cloned into a pTZ57R/T-vector, transferred to an expression vector pET-23a (+) and expressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme purified to 19.37-fold had a molecular weight of 30 kDa (SDS-PAGE analysis). Scanning Electron Microscopy (SEM) revealed changes in the surface morphology of native and treated PHA films. Further, changes in molecular vibrations were confirmed by Fourier Transform Infrared Spectroscopy.