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Applied and Environmental Microbiology, April 2009, p. 2148-2157, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02103-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Novel Coprinopsis cinerea Polyesterase That Hydrolyzes Cutin and Suberin{triangledown} ,{dagger}

Hanna Kontkanen, Ann Westerholm-Parvinen, Markku Saloheimo, Michael Bailey, Marjaana Rättö, Ismo Mattila, Marzia Mohsina,{ddagger} Nisse Kalkkinen,§ Tiina Nakari-Setälä, and Johanna Buchert*

VTT, P.O. Box 1000, FI-02044 Espoo, Finland

Received 11 September 2008/ Accepted 23 January 2009

Three cutinase gene-like genes from the basidiomycete Coprinopsis cinerea (Coprinus cinereus) found with a similarity search were cloned and expressed in Trichoderma reesei under the control of an inducible cbh1 promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenylbutyrate, used as a model substrate. The most promising transformant of the cutinase CC1G_09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter–l purified protein. The expressed cutinase (CcCUT1) was purified to homogeneity by immobilized metal affinity chromatography exploiting a C-terminal His tag. The N terminus of the enzyme was found to be blocked. The molecular mass of the purified enzyme was determined to be around 18.8 kDa by mass spectrometry. CcCUT1 had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUT1 had optimal activity between pH 7 and 8 but retained activity over a wide pH range. The enzyme retained 80% of its activity after 20 h of incubation at 50°C, but residual activity decreased sharply at 60°C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.

* Corresponding author. Mailing address: VTT, P.O. Box 1000, FI-02044 Espoo, Finland. Phone: 358 20 722 5146. Fax: 358 20 722 7071. E-mail: johanna.buchert{at}vtt.fi

{triangledown} Published ahead of print on 6 February 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: University of Helsinki, Faculty of Veterinary Medicine, Agnes Sjöberginkatu 2, 00014 University of Helsinki, Finland.

§ Present address: Protein Chemistry Research Group and Core Facility, Institute of Biotechnology, P.O. Box 65, 00014 University of Helsinki, Finland.

Applied and Environmental Microbiology, April 2009, p. 2148-2157, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.02103-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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