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 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 30 min at 50 degrees C (optimum temperature 25 degrees C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain p-nitrophenyl esters of rho-nitrophenylcaprylate (C8), rho-nitrophenyllaurate (C12) and rho-nitrophenylmyristate (C14), with the highest catalytic efficiency, k(cat)/K-m of 7.7 +/- 0.7 mM(-1) s(-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. (C) 2014 Elsevier B.V. All rights reserved.