Short Communication Gene cloning and molecular characterization of the Talaromyces thermophilus lipase Catalyzed efficient hydrolysis and synthesis of esters Ines Belhaj-Ben Romdhane a , Fakher Frikha b , Inès Maalej-Achouri a , Ali Gargouri a , Hafedh Belghith a, ⁎ a Laboratoire de Valorisation de la Biomasse et Production des Protéines chez les Eucaryotes Centre de Biotechnologies de Sfax, BP “1177” 3018 Sfax, University of Sfax, Tunisia b Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route Soukra, Université de Sfax, Tunisia abstract article info Article history: Accepted 22 November 2011 Available online 9 December 2011 Received by A.J. van Wijnen Keywords: Lipase Talaromyces thermophilus Cloning gene Promoter sequence Transesterification A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously iso- lated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple ap- plications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response El- ement (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expres- sion was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic effi- ciency even reached 91.6% from Waste Cooking Oil triglycerides. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Lipases are triacylglycerol-hydrolyzing enzymes whose activity depends on the occurrence of the oil–water interface. Apart from their hydrolysis properties in aqueous medium, lipases in non- aqueous medium are efficient catalysts in the kinetic resolution of chiral compounds, synthesis of esters and peptides, and preparation of biodiesel by trans-esterification (Alatorre-Santamarίa et al., 2009; Yang et al., 2009). Lipases are also extensively used in detergents for- mulations and food processing (Pandey et al., 1999) as well as in bio- logical degradation of fatty waste (Hansan et al., 2006). The development of industrial applications has lead to increased interest in lipase structure and functional studies of many cloned li- pases genes from microorganisms (Pleiss et al., 2000). Lipases are, in general, highly variable in size and the sequence similarity be- tween them is limited to short spans located around the active-site residues. However, the three-dimensional structures of lipases, in their cores, share a common fold motif, known as α/β hydrolase fold (Ollis et al., 1992). This fold consists of an eight-stranded, mostly parallel β sheet flanked by six α helices, with a catalytic triad (Ser/ Asp/Cys-His-Asp/Glu). One of the most conserved features of the α/ β-hydrolase enzymes is the nucleophile elbow, a sharp γ turn con- taining the nucleophilic serine residue, positioned between a β strand and the following α helix (Schrag and Cygler, 1997). Above the ser- ine, a hydrophobic cleft is present or formed after activation of the en- zyme (Panaiotov and Verger, 2000). Microbial lipases have attracted particular attention due to their high production potential, diverse properties and easy availability. The varied applications of these enzymes necessitate continued search of new lipase producers that can expand the assortment of specificities and feasible operating conditions. Lipases from extreme environments have recently attracted attention because they are heat/cold-tolerant, show organic solvents tolerance and special cata- lytic activities (Kiran et al., 2008). In previous studies, we isolated a thermophilic strain of Talaromyces thermophilus that is able to secrete a high level of lipolytic activity when it has grown on wheat bran as the only carbon source. Biochemical characterization of this novel pu- rified lipase (TTL) revealed that its properties differ markedly from those of other reported lipases because of its ability to remain stable and active under drastic conditions, including the presence of deter- gent, alkaline pH, and temperatures as high as 60 °C (Belhaj-Ben Romdhane et al., 2010a). This lipase had also an efficient potential in catalytic esterification (Belhaj-Ben Romdhane et al., 2010b). Here we report the cloning and sequencing of this lipase gene with its upstream regulatory region. Peculiar characteristics of the se- quence of responsive cis regulatory elements are given and discussed Gene 494 (2012) 112–118 Abbreviations: TTL, Talaromyces thermophilus lipase; PCR, Polymerase Chain Reac- tion; ORE, Oleate Response Element; FARE, Fatty acids Responsis Element; SSC, Saline Sodium Citrate; TC4, Tributyrin; TC8, Trioctanoin; GA, Gum Arabic; FAMEs, Fatty Acid Methyl Esters; RMSD, Root Mean Squared Deviation; TRANSFACT, Transcription Factor Binding Sites Database; WCO, Waste Cooking Oil. ⁎ Corresponding author. Tel./fax: + 216 74874449. E-mail address: hafeth.belghith@cbs.rnrt.tn (H. Belghith). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.11.059 Contents lists available at SciVerse ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene