Monday, July 25, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
Cutinase (EC 3.1.1.74) is a carboxylic ester hydrolases that is capable of degrading cutin polymers of plant cell wall. Cutinases can be considered as a link between esterases and lipases as they efficiently hydrolyze soluble esters and emulsified triacylglycerols, and shows activity towards variety of soluble synthetic esters, insoluble triglycerides, synthetic fibers (polyethylene terephthalate fibers) and plastics (poly- caprolactone) etc. Due to such unique properties, it is emerging as an important enzyme in industrial biotechnological applications. In the present investigation we have cloned and expressed two cutin hydrolyzing genes from Thermobifida fusca in E.coli BL21 (DE3). These recombinant enzymes were purified by IMAC and characterized. The factors influencing the product yields of these processes like temperature, ionic strength, pH, substrate specificity, effect of metal ions and stability in surfactant and organic solvents were investigated. We have studied the combined denaturing effect of pH and temperature on cutinase using response surface methodology. The thermodynamic parameters for cutinase deactivation have also been evaluated. The study showed these two enzymes are 93% identical in amino acid sequences. The recombinant enzymes have higher sp. activity in comparison to wild Thermobifida fusca enzyme. Initial biochemical characterization showed that both enzymes have similar temperature dependence profiles, thermostability and display higher temperature optimum with greater thermostability, higher surfactant and organic solvent tolerance compared to reported fungal and other cutinases. In conclusion, Thermobifida fusca cutinase would be a promising alternative to existing cutinases of various sources in biotechnological applications in industries.