BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Cloning and characterization of a novel acidic cutinase from Sirococcus conigenus Antti Nyyssölä & Ville Pihlajaniemi & Mari Häkkinen & Hanna Kontkanen & Markku Saloheimo & Tiina Nakari-Setälä Received: 3 June 2013 /Revised: 23 September 2013 /Accepted: 26 September 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract A cutinase gene (ScCut1 ) was amplified by PCR from the genomic DNA of the ascomycetous plant pathogen Sirococcous conigenus VTT D-04989 using degenerate primers designed on the basis of conserved segments of known cutinases and cutinase-like enzymes. No introns or N - or O - glycosylation sites could be detected by analysis of the ScCut1 gene sequence. The alignment of ScCut1 with other fungal cutinases indicated that ScCut1 contained the conserved motif G-Y-S-Q-G surrounding the active site serine as well as the aspartic acid and histidine residues of the cutinase active site. The gene was expressed in Pichia pastoris , and the recombinantly produced ScCut1 enzyme was purified to ho- mogeneity by immobilized metal affinity chromatography exploiting a C-terminal His-tag translationally fused to the protein. The purified ScCut1 exhibited activity at acidic pH. The K m and V max values determined for p NP-butyrate esterase activity at pH 4.5 were 1.7 mM and 740 nkat mg -1 , respective- ly. Maximal activities were determined at between pH 4.7 and 5.2 and at between pH 4.1 and 4.6 with p NP-butyrate and tritiated cutin as the substrates, respectively. With both sub- strates, the enzyme was active over a broad pH range (between pH 3.0 and 7.5). Activity could still be detected at pH 3.0 both with tritiated cutin and with p -nitrophenyl butyrate (relative activity of 25 %) as the substrates. ScCut1 showed activity towards shorter (C2 to C3) fatty acid esters of p -nitrophenol than towards longer ones. Circular dichroism analysis sug- gested that the denaturation of ScCut1 by heating the protein sample to 80 °C was to a great extent reversible. Keywords Cutinase . Esterase . Cutin . Suberin . Polyester Introduction Cutin and suberin are biopolymers present in the protective layers on the surfaces of plants. These polymers act mainly as barriers for water and other small compounds. It has been estimated that cutin is, after cellulose and lignin, the third most abundant biopolymer in nature and the major lipid plant polymer. Cutin is present in significant amounts for example in berries, cereals, fruits, and vegetables, whereas suberin is found in tree bark, cork, and roots (Kolattukudy 1980; Holloway 1984; Heredia et al. 2000). Cutin is composed of variably substituted fatty acids interlinked with ester bonds. The typical structural components of cutin are hydroxyl and epoxy substituted ω-hydroxy fatty acids with C16 and C18 carbon chains (Kolattukudy 2001). Suberin is composed of a polyphenolic domain and of a polyester domain similar to cutin. However, fatty acids with longer carbon chains may be present in suberin and the form of cross-linking may differ from that of cutin (Bernards 2002). Cutinases (EC 3.1.1.74) are extracellular enzymes, which catalyze the hydrolysis of the ester bonds of cutin, suberin, lipids, and waxes (Longhi and Cambillau 1999). All biochem- ically well-characterized cutinases are serine esterases, con- taining the S-H-D triad similar to serine proteases and several lipases (Carvalho et al. 1998). Soluble esters are also hydro- lyzed by cutinases (Purdy and Kolattukudy 1975), suggesting A. Nyyssölä (*) : V. Pihlajaniemi : M. Häkkinen : H. Kontkanen : M. Saloheimo : T. Nakari-Setälä (*) VTT Biotechnology, P.O. Box 1000, 02044 Espoo, Finland e-mail: Antti.Nyyssola@vtt.fi e-mail: Tiina.Nakari-Setala@vtt.fi Present Address: V. Pihlajaniemi Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, P.O. Box 16100, 00076 Espoo, Finland Present Address: H. Kontkanen Valio Ltd., R&D Center, P.O. Box 30, 00039 Helsinki, Finland Appl Microbiol Biotechnol DOI 10.1007/s00253-013-5293-z