Seman_2014_J.Biotechnol_184C_219

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL-1 wet cell weight) with a cutinase production of 3800mgL-1 and an activity of 434UmL-1 were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28 degrees C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50 degrees C (optimum temperature 25 degrees C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain rho-nitrophenyl esters of rho-nitrophenylcaprylate (C8), rho-nitrophenyllaurate (C12) and rho-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7+/-0.7mM-1s-1 for rho-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.