Process Biochemistry 46 (2011) 888–893 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Purification and biochemical characterization of a novel thermoactive fungal pectate lyase from Penicillium occitanis Naourez Damak a , Noomen Hadj-Taieb a , Estelle Bonnin b , Abir Ben Bacha c , Ali Gargouri a,∗ a Laboratoire de Génétique Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, BP «1177», University of Sfax, 3018 Sfax, Tunisia b Unité Biopolymères, Interactions, Assemblages (BIA), INRA, BP 71627, F-44300 Nantes, France c Unité de Service Commun à la Recherche, Faculté des Sciences de Sfax, 3038 Sfax, Tunisia article info Article history: Received 30 April 2010 Received in revised form 22 December 2010 Accepted 23 December 2010 Keywords: Penicillium Pectate lyase Pectin Thermoactivity Alkalophilicity abstract We purified a novel, thermoactive pectate lyase (Pel), designated Pel1, from the hyperpectinolytic mutant (CT1) of Penicillium occitanis using single-step, anion-exchange chromatography. The purified Pel1 had a pI of 6.5 and a molecular mass of approximately 39 kDa as estimated by SDS-PAGE. The activity of Pel1 was assayed in the presence of several divalent cations at different concentrations. Although we noted that the tested divalent cations are required for maximum activity, we found that none of them could substitute for the effects of 2 mM Ca 2+ . Using three different carbon sources, our results showed that the highest level of Pel1 activity (434 U/ml) was obtained in a medium supplemented with apple pectin. With polygalacturonic acid, Pel1 exhibited a K m of 0.0199 mg/ml and a V max of 17.19 mol/min/mg at pH 7.0. However, at pH 9.0, the K m and V max values were 0.0136 mg/ml and 19.23 mol/min/mg, respectively. The optimum temperature and pH value required for maximal enzymatic activity were found to be 60 ◦ C and pH 9.0, respectively. At this pH, the half-life time stability at 60 ◦ C was measured to be 16.4 min. The novel Pel1 is very thermally stable and presents alkalophilic parameters; such features are rarely exhibited by fungal Pels. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction The plant cell wall is organized as a complex network composed of polysaccharides such as cellulose, hemicellulose, and pectin. Pectins are complex heteropolysaccharides consist- ing of homogalacturonan (“smooth,” -(1 → 4)-linked units that are partially d-GalAp-˛-(1 → 2) methylesterified) and rhamno- galacturonan (“hairy,” GalAp--(1 → 2)-Rhap--(1 → 4)-GalAp- (1 → 2)-Rhap) regions [1,2]. In homogalacturonan, some of the hydroxyl groups on the C2 and C3 positions may be acetylated [3]. In rhamnogalacturonan, the neutral sugar side chains, contain- ing mainly l-arabinose, d-galactose, and d-xylose, are covalently attached to the main chain at the C1 and C2 positions [4]. Pectin degradation requires the combined action of several enzymes that have been classified into three groups, namely methylesterases, depolymerases, and debranching enzymes. The methylesterases remove methoxyl groups from pectin. The depoly- merases (hydrolases and lyases) cleave bonds between the galacturonate units in linear galacturonans. The debranching enzymes cut at the branch points (the “hairy” regions). ∗ Corresponding author. Tel.: +216 74 874 449; fax: +216 74 874 449. E-mail address: faouzi.gargouri@cbs.rnrt.tn (A. Gargouri). Pectate lyases (Pels) catalyze the random cleavage of internal -1,4-linkages of polygalacturonate through a trans-eliminative mechanism, thereby generating 4,5-unsaturated oligogalactur- onates [5]. The activity of Pels was first discovered by Starr and Moran [6] in cultures of Erwinia carotovora and Bacillus polymyxa bacteria. Further studies reported that other bacteria such as Pseu- domonas [7], Streptomyces [8], and Thermoanaerobacter [9] are able to produce Pels. The genes of Pels have been cloned and sequenced, and their proteins have been purified from microor- ganisms [10–12]. However, Pel activity has been rarely reported in fungi, an example being Fusarium [13]. The fungal Pels, such as those in Fusarium solani and Aspergillus niger, have been expressed in Pichia pastoris, Escherichia coli, and Bacillus followed by purifica- tion [14–18]. Pels share sequence similarities that range between 20% and 90% and generally fall into two subclasses, depending upon the number of disulfide bonds and the length of the final cleavage product [19,20]. The enzymatic degradation of pectin is crucial in several indus- trial and biotechnological processes, such as those involved with the fruit juice [21], textile, and paper treatment industries [4]. The endo-Pel of Bacillus pumilus BK2 has a high isoelectric point (pI) and a high optimum pH, which are highly valued for a number of industrial applications such as the bioscouring of cotton [22]. Despite the promising properties and attributes of Pels and the sheer volume of work dedicated to these enzymes, to date and to 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.12.014