title: High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions.
abstract: 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.
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
authors.forename: W M K Wan
authors.initials: WM
authors.lastname: Seman
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
authors.forename: S A
authors.initials: SA
authors.lastname: Bakar
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
authors.forename: N A
authors.initials: NA
authors.lastname: Bukhari
authors.affiliation: Syarikat Bumi Sains, 16, 1(st) Floor,Taman City, Off Jalan Kuching, 51200 Kuala Lumpur, Malaysia.
authors.forename: S M
authors.initials: SM
authors.lastname: Gaspar
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.
authors.forename: R
authors.initials: R
authors.lastname: Othman
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; Malaysia Genome Institute, Ministry of Science, Technology and Innovation, Jalan Bangi, 43000 Kajang, Selangor, Malaysia.
authors.forename: S
authors.initials: S
authors.lastname: Nathan
authors.affiliation: Malaysia Genome Institute, Ministry of Science, Technology and Innovation, Jalan Bangi, 43000 Kajang, Selangor, Malaysia; Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.
authors.forename: N M
authors.initials: NM
authors.lastname: Mahadi
authors.affiliation: Department of Chemical and Process Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
authors.forename: J
authors.initials: J
authors.lastname: Jahim
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
authors.forename: A M A
authors.initials: AM
authors.lastname: Murad
authors.affiliation: School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. Electronic address: fabyff@ukm.edu.my.
authors.forename: F D Abu
authors.initials: FD
authors.lastname: Bakar
mesh_terms: D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D020081:Phyllachorales / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000254:growth & development; D011093:Polyethylene Terephthalates / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature
chemical_list: D011093:Polyethylene Terephthalates; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D002265:Carboxylic Ester Hydrolases; C019952:cutinase
keywords: Colletotrichum gloeosporioides; Polyethylene terephthalate; Surfactants; Thermostability; ρ-Nitrophenylesters