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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23188 43419 2014 10 PDF Available
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 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.

Keywords
DO, dissolved oxygen; doT, dissolved oxygen tension; Mut+, methanol utilisation positive; PTM1, Pichia trace metals 1; ρNPA, ρ-nitrophenylacetate (C2); ρNPP, ρ-nitrophenyl proprionate (C3); ρNPB, ρ-nitrophenylbutyrate (C4); ρNPV, ρ-nitrophenylvalerate (C5
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High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 184, 20 August 2014, Pages 219–228
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us