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

J Biotechnol. 2014 Aug 20;184:219-28. doi: 10.1016/j.jbiotec.2014.05.034. Epub 2014 Jun 6.


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°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°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±0.7mM(-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.

Keywords: Colletotrichum gloeosporioides; Polyethylene terephthalate; Surfactants; Thermostability; ρ-Nitrophenylesters.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Batch Cell Culture Techniques / methods*
  • Bioreactors
  • Carboxylic Ester Hydrolases / biosynthesis*
  • Carboxylic Ester Hydrolases / chemistry
  • Carboxylic Ester Hydrolases / metabolism
  • Hydrogen-Ion Concentration
  • Phyllachorales / enzymology*
  • Phyllachorales / genetics
  • Pichia / genetics
  • Pichia / growth & development
  • Polyethylene Terephthalates / chemistry
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Surface-Active Agents / chemistry
  • Temperature


  • Polyethylene Terephthalates
  • Recombinant Proteins
  • Surface-Active Agents
  • Carboxylic Ester Hydrolases
  • cutinase