Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre

Abstract

An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condition has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chromatography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphatepolyacrylamide gel electrophoresis analysis with a molecular mass corresponding to 75.5 kDa. The Km and kcat values of the PG were 2.7 mg/mL and 2.23 × 10 3 s −1 , respectively, using citrus polygalacturonic acid as the substrate. The optimum pH of the purified PG was 5.0 and it does not loose activity appreciably if left for 24 hours in the pH range from 5.0 to 12.0. The optimum temperature of purified enzyme was 50• C and the enzyme does not loose activity below 30• C if exposed for two hours. The purified enzyme showed complete inhibition with 1 mM Ag + , Hg 2+ and KMnO4, while it was stimulated to some extent by Co 2+ . The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

“…Similar pH optimum of 4.0 has been reported for an exo-PG purified from Aspergillus sojae and Paecilomyces variotii de Lima Damasio et al 2010). Previous reports show that majority of fungal PGs have their pH optima in acidic range (Kester and Visser 1990;Polizeli et al 1991;Niture and Pant 2004;Yadav et al 2012). However, pH stability range of the enzyme was wider (3.0-11.0) as compared to fungal PGs reported from A. niger (Kant et al 2013).…”

“…3d). Similar temperature optimum has been reported for PG from R. oryzae (Yadav et al 2012). Several fungal PGs exhibit temperature optima between 40 and 60°C (Devi and Rao 1996;Kaur et al 2004;Tari et al 2008;Thakur et al 2010).…”

“…Ion-exchange chromatography resulted in 15.28-fold purification with 1.2% yield and 33.47 U/mg of specific activity (Table 1). 16-fold purification with 1.9% yield has been reported for exo-PG from Rhizopus oryzae (Yadav et al 2012).…”

“…An exo-PG from A. tubingensis has K m value of 3.2 mg/mL (Kester et al 1996). The K m values of most of the microbial PGs are in the range of 0.1-5.0 (Polizeli et al 1991;Zhang et al 1999;Niture et al 2001;Singh and Rao 2002;Thakur et al 2010a, b;Yadav et al 2012;Martins et al 2013;Chen et al 2014). An exo-PG from the fungus Penicillium frequentans has very low K m value of 0.059 (Barense et al 2001) while PG from pathogenic fungus Ustilago maydis revealed comparatively very high K m of 57.84 (Castruita-Domínguez et al 2014).…”

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

“…3C and G). The optimal temperature of NfPG4 is higher than those of the exo-polygalacturonases of Penicillium viridicatum RFC3 (60 • C) [23], A. sojae (55 • C) [24], Rhizopus oryzae MTCC 1987 (50 • C) [25], and the exo-polygalacturonase NfPG3 of N. fischeri P1 (60 • C)(to be published elsewhere), respectively, equal to that of an exo-polygalacturonase from Paecilomyces variotii (65 • C) [22] ( Table 2). The optimal temperature of the endopolygalacturonase NfPG5 is higher than any other characterized fungal endopolygalacturonases such as the endopolygalacturonases from A. tubingensis (60 • C) [26], A. niger JL-15 (50 • C) [27], Achaetomium sp.…”