High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5 L bioreactor (2.0 L 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 (320 g L−1 wet cell weight) with a cutinase production of 3800 mg L−1 and an activity of 434 U mL−1 were achieved 24 h after induction with methanol in basal salt medium (at pH 5 and 28 °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 30 min at 50 °C (optimum temperature 25 °C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7 ± 0.7 mM−1 s−1 for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.