Journal of Molecular Catalysis B: Enzymatic 127 (2016) 56–66 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic j ourna l ho me pa g e: www.elsevier.com/locate/molcatb Catalytic properties of a highly thermoactive polygalacturonase from the mesophilic fungus Penicillium occitanis and use in juice clarification Hajer Tounsi a , Azza Hadj Sassi a , Zamen Ben Romdhane a , Marwa Lajnef a , Jean-William Dupuy b , Delphine Lapaillerie b , Anne-Marie Lomenech b , Marc Bonneu b , Ali Gargouri a,∗ , Noomen Hadj-Taieb a a Laboratoire de Biotechnologie Moléculaire des Eucaryotes, Centre de Biotechnologie de Sfax, University of Sfax, BP1177, 3018 Sfax, Tunisia b Centre de Génomique Fonctionnelle, Plateforme Protéome, Université de Bordeaux, F-33000 Bordeaux, France a r t i c l e i n f o Article history: Received 20 November 2015 Received in revised form 20 February 2016 Accepted 21 February 2016 Available online 24 February 2016 Keywords: Penicillium occitanis Polygalacturonase Specific activity Thermoactivity Juice extraction a b s t r a c t A new polygalacturonase (PG1) is purified to homogeneity from the hyper-pectinolytic mutant (CT1) of Penicillium occitanis by using two chromatographic steps. The purified PG1 exhibited a very high specific activity toward PGA, namely 57533.2 U/mg of protein and a high optimal temperature, 70 ◦ C. In our knowledge, it is one of the highest temperature optima described till now, even among those reported for thermophilic fungi; recalling here that our fungus is a mesophilic one. The N-terminal sequence was almost identical to that deduced from the previously cloned pga1 gene. The mass spectrometry analysis of PG1 further confirmed its belonging to the pga1 gene. As the peptide sequence of the pga1 bears the signature of endopolygalacturonases, we brought in this work evidences that it belongs to enzymes that hydrolyze pectin by endo-fashion. Indeed, during the hydrolysis of polygalacturonic acid by PG1, the viscosity drops very quickly while reducing sugars were released very slowly. In addition, thin layer chromatography showed that mainly oligosaccharides were released from PGA, namely tetra and tri- galacturonic acids. The purified PG1 was able to enhance the clarification of citrus juice. Considering all these properties, this novel fungal would be an interesting biocatalyst for future application in fruit and vegetable transformation. © 2016 Elsevier B.V. All rights reserved. 1. Introduction The biological decomposition of pectin is an important process in biomass degradation. In nature, pectin is degraded by enzymatic systems produced by a wide variety of saprophytic and phyto- pathogenic micro-organisms, including bacteria, yeasts and fungi. The cell wall polysaccharides of plants are composed of cellu- lose, hemicellulose and pectin. Cellulose is the main constituent, followed by hemicelluloses which are both characterized by a backbone of 1,4-linked sugars. Pectins are the third structural polysaccharide of plant cell walls and consist mainly of 1,4-linked d-galacturonic acids that are partially methyl or acetyl esterified at various degrees. This homogalacturonan is linked to two types of ramified regions, called also hairy regions that contain a small num- ber of rhamnose residues in the main chain and arabinose, galactose ∗ Corresponding author. Fax: +216 74 874 449. E-mail address: Faouzi.gargouri@cbs.rnrt.tn (A. Gargouri). and xylose residues on side chains. The degradation of pectin can be achieved by the combined and synergistic action of several pecti- nolytic enzymes. The pectinases, acting on the homogalacturonan smooth region, are classified into three groups: 1-esterases: methyl and acetyl esterases (respectively EC 3.1.1.11 and EC 3.1.1.6), which remove methyl or acetyl groups from pectin, 2-hydrolases: composed essentially of polygalacturonases (EC 3.2.1.15) which hydrolyze the osidic linkage in pectin or pectate (polygalacturonic acid or PGA); it encloses also enzymes acting on rhamnogalac- turonan or RG regions, such as the rhamnogalacturonases which cleave the linkage between galacturonic acid and rhamnose in the RG backbone; 3- Lyases: pectin and pectate lyases (respectively EC 4.2.2.10 and EC 4.2.2.2), which cleave the bonds in pectin and pec- tate respectively by beta-elimination [1]. A fourth heterogeneous group encompasses a multitude of debranching enzymes that act on the hairy regions, such as rhamnnosidases, -galactosidase, ara- binanase, arabinofuranosidase and other enzymes [1,2]. Polygalacturonases (PGs) have been most extensively studied among other families of pectinolytic enzymes [1] and all PGs belong http://dx.doi.org/10.1016/j.molcatb.2016.02.012 1381-1177/© 2016 Elsevier B.V. All rights reserved.