TY - JOUR
T1 - Novel Coprinopsis cinerea polyesterase that hydrolyzes cutin and suberin
AU - Kontkanen, Hanna
AU - Westerholm-Parvinen, Ann
AU - Saloheimo, Markku
AU - Bailey, Michael
AU - Rättö, Marjaana
AU - Mattila, Ismo
AU - Mohsina, Marzia
AU - Kalkkinen, Nisse
AU - Nakari-Setälä, Tiina
AU - Buchert, Johanna
PY - 2009/4
Y1 - 2009/4
N2 - 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.
AB - 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.
KW - Agaricales/enzymology
KW - Butyrates/metabolism
KW - Chromatography, Affinity
KW - Cloning, Molecular
KW - DNA, Fungal/chemistry
KW - Enzyme Stability
KW - Gene Expression
KW - Hydrogen-Ion Concentration
KW - Hydrolases/chemistry
KW - Kinetics
KW - Lipids
KW - Membrane Lipids/metabolism
KW - Molecular Sequence Data
KW - Molecular Weight
KW - Sequence Analysis, DNA
KW - Substrate Specificity
KW - Temperature
KW - Trichoderma/genetics
U2 - 10.1128/AEM.02103-08
DO - 10.1128/AEM.02103-08
M3 - Journal article
C2 - 19201950
SN - 0099-2240
VL - 75
SP - 2148
EP - 2157
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 7
ER -