Two thermophilic fungal pectinases from Neosartorya fischeri P1: Gene cloning, expression, and biochemical characterization

摘要

In addition to cellulose and hemicellulose, pectin is an important renewable resource for biorefinery. Endo- and exo-polygalacturonases are two essential enzymes required for complete deconstruction of pectin into simple and fermentable sugars. In biorefinery, thermophilic pectinases are more appealing than their mesophilic counterparts. Two pectinase genes encoding a putative exo-polygalacturonase (NfPG4) and a putative endopolygalacturonase (NfPG5), respectively, were thus cloned from a thermophilic filamentous fungus Neosartorya fischeri P1 and successfully expressed in Pichia pastoris. NfPG4 and NfPG5 share 79% and 58% identity, respectively, with their closest characterized homologs from Aspergillus riibingensis and Achaetomium sp. Xz-8. They are both acidic and thermophilic, with maximum activities at pH 3.5 and 65 degrees C for NfPG4 and pH 4.5 and 70 degrees C for NfPG5, respectively. In comparison with other characterized thermophilic polygalacturonases, NfPG5 is the most thermophilic fungal endopolygalacturonase. Both enzymes have excellent stability at 55 degrees C and over a broad pH range (pH 3.0-8.0 for NfPG4 and pH 2.0-11.0 for NfPG5), and are strongly resistant to many metal ions and chemical reagents. NfPG5 has a typical endo-mode of action on polygalacturonic acid, releasing galacturonic acid, di-galacturonic acid, and tri-galacturonic acid. In contrast, NfPG4 only releases galacturonic acid as the end hydrolysis product, typified by exo-polygalacturonases. NfPG4 has a specific activity of 700.9 U/mg, higher than most characterized exo-polygalacturonases. NfPG5 is also a robust polygalacturonase with a specific activity of 3630.4 U/mg. These superior properties make the two polygalacturonases interesting candidates for industrial applications with a preference for thermophilic pectinases. (C) 2015 Elsevier B.V. All rights reserved.