Enzyme and Microbial Technology 52 (2013) 272–278 Contents lists available at SciVerse ScienceDirect Enzyme and Microbial Technology j our na l ho me p age: www.elsevier.com/locate/emt Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88 Antti Nyyssölä a,∗ , Ville Pihlajaniemi a,2 , Riikka Järvinen b , Saara Mikander a,1 , Hanna Kontkanen a,1 , Kristiina Kruus a , Heikki Kallio b , Johanna Buchert a a VTT Biotechnology, P.O. Box 1000, FIN-02044 VTT, Finland b Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland a r t i c l e i n f o Article history: Received 20 November 2012 Received in revised form 8 January 2013 Accepted 9 January 2013 Keywords: Cutin Polyester Cutinase Esterase Plant pathogen Fungi a b s t r a c t Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC–MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5. © 2013 Elsevier Inc. All rights reserved. 1. Introduction The cuticle is a layer that protects the aerial parts of plants from microbial infections, desiccation and loss of solutes [1,2]. The major constituent of the cuticle is cutin, which is composed of vari- ably substituted fatty acids interlinked with ester bonds. The main structural components of cutin are hydroxyl and epoxy substituted -hydroxy fatty acids with C16 and C18 carbon chain lengths [3]. Many micro-organisms produce cutinases (EC 3.1.1.74), which catalyze the hydrolysis of the ester bonds of cutin. Cutinases are especially common for fungal plant pathogens (e.g. Fusaria, Botry- tis cineria and Venturia inaequalis) and it is believed that cutinase catalyzed degradation of the cuticular layer enables the fungi to penetrate the surfaces of plants [4–6]. Prokaryotic cutinase pro- ducers such as Thermobifida fusca [7] and various Pseudomonas strains [8,9] are also known. All characterized cutinases are ser- ine esterases, which contain the catalytic triad Ser, His, and Asp, common also for serine proteases and lipases [10]. The pH optima ∗ Corresponding author. Tel.: +358 40 350 3554; fax: +358 20 722 7071. E-mail address: antti.nyyssola@vtt.fi (A. Nyyssölä). 1 Current address: Valio Ltd. Research and Development Center, FIN-00039 Valio, Finland. 2 Current address: Aalto University, School of Chemical Technology, Department of Biotechnology and Chemical Technology, P.O. Box 16100, 00076 Aalto, Finland. reported for the cutinases are almost exclusively either in the neu- tral or alkaline range. A variety of applications have been suggested for cutinolytic enzymes. Cutinases could be used in detergents for dishwashing and laundry applications. For use in the textile industry, a bioscour- ing method utilizing cutinases for the removal of the waxy layer present in cotton has been developed [11]. Cutinases have also been tested for modification of the surfaces of polyester fibers [12]. The use of cutinases for the detoxification of feed products contami- nated by the heat-stable mycotoxin zearalenone has been patented [13]. Processing of fruits, vegetables and berries generates millions of tons of acidic by-products annually. These cutin rich materials are mainly disposed of as waste or used as animal feed [14–16]. Cutinases could therefore be used in facilitating the release of bioac- tive compounds from these materials. Many cuticular fatty acids show interesting functionalities, which could be utilized in the pro- duction of for example lubricants and binders [17]. In addition, cutinases have also been suggested for use in combination with other hydrolytic enzymes, such as cellulases, for the degradation of plant materials [18]. Despite their wide potential, the number of applications for known cutinases is limited, since their cutinolytic activities are either poor or nonexistent at low pH. In particular, plant derived materials are in many cases acidic. Many environmental applica- tions for cutinases would also require functionality at low pH. In the 0141-0229/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.enzmictec.2013.01.005