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

W. M K Wan Seman, S. A. Bakar, N. A. Bukhari, S. M. Gaspar, Roohaida Othman, Sheila Nathan, N. M. Mahadi, Jamaliah Md Jahim, Abdul Munir Abd. Murad, Farah Diba Abu Bakar

Research output: Contribution to journalArticle

16 Citations (Scopus)

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-1s-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.

Original languageEnglish
Pages (from-to)219-228
Number of pages10
JournalJournal of Biotechnology
Volume184
DOIs
Publication statusPublished - 20 Aug 2014

Fingerprint

Phyllachorales
Pichia
Bioreactors
Polyethylene terephthalates
Polyesters
Esters
Methanol
Molecular weight
Salts
Temperature
Substrates
Polyethylene Terephthalates
Experiments
Complementary DNA
Cell Count
Molecular Weight
cutinase
Weights and Measures

Keywords

  • Colletotrichum gloeosporioides
  • Polyethylene terephthalate
  • Surfactants
  • Thermostability
  • ρ-Nitrophenylesters

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

Cite this

High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions. / Seman, W. M K Wan; Bakar, S. A.; Bukhari, N. A.; Gaspar, S. M.; Othman, Roohaida; Nathan, Sheila; Mahadi, N. M.; Md Jahim, Jamaliah; Abd. Murad, Abdul Munir; Abu Bakar, Farah Diba.

In: Journal of Biotechnology, Vol. 184, 20.08.2014, p. 219-228.

Research output: Contribution to journalArticle

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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-1s-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.",
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AU - Bakar, S. A.

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AU - Gaspar, S. M.

AU - Othman, Roohaida

AU - Nathan, Sheila

AU - Mahadi, N. M.

AU - Md Jahim, Jamaliah

AU - Abd. Murad, Abdul Munir

AU - Abu Bakar, Farah Diba

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AB - 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-1s-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.

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