Novel Coprinopsis cinerea Polyesterase that hydrolyzes cutin and Suberin

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

Research output: Contribution to journalArticleScientificpeer-review

34 Citations (Scopus)

Abstract

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 cbhl promoter. The selected transformants of all three polyesterase constructs showed activity with p-nitrophenyl-butyrate, used as a model substrate. The most promising transformant of the cutinase CCIG-09668.1 gene construct was cultivated in a laboratory fermentor, with a production yield of 1.4 g liter-1 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. CcCUTl had higher activity on shorter (C2 to C10) fatty acid esters of p-nitrophenol than on longer ones, and it also exhibited lipase activity. CcCUTl 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.

Original languageEnglish
Pages (from-to)2148-2157
Number of pages10
JournalApplied and Environmental Microbiology
Volume75
Issue number7
DOIs
Publication statusPublished - 1 Apr 2009
MoE publication typeA1 Journal article-refereed

Keywords

  • polyesterases
  • Coprinopsis cinerea
  • hydrolyzing
  • cutin
  • cutinase
  • suberin

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