Novel Coprinopsis cinerea polyesterase that hydrolyzes cutin and suberin.
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 (C(2) to C(10)) 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 degrees C, but residual activity decreased sharply at 60 degrees C. Microscopic analyses and determination of released hydrolysis products showed that the enzyme was able to depolymerize apple cutin and birch outer bark suberin.
Kontkanen
Hanna
H
VTT, P.O. Box 1000, FI-02044 Espoo, Finland.
Westerholm-Parvinen
Ann
A
Saloheimo
Markku
M
Bailey
Michael
M
Rättö
Marjaana
M
Mattila
Ismo
I
Mohsina
Marzia
M
Kalkkinen
Nisse
N
Nakari-Setälä
Tiina
T
Buchert
Johanna
J
D000363:Agaricales / Q000201:enzymology* / Q000235:genetics*; D002087:Butyrates / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids; D008563:Membrane Lipids / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000235:genetics
D002087:Butyrates; D004271:DNA, Fungal; D008055:Lipids; D008563:Membrane Lipids; C033592:4-nitrophenyl butyrate; C000521:cutin; C065875:suberin; D006867:Hydrolases