Author’s copy Biologia 67/6: 1069—1074, 2012 Section Cellular and Molecular Biology DOI: 10.2478/s11756-012-0122-x Purification and characterization of an exo-polygalacturonase secreted by Rhizopus oryzae MTCC 1987 and its role in retting of Crotalaria juncea fibre Sangeeta Yadav, Gautam Anand, Amit K. Dubey & Dinesh Yadav Department of Biotechnology, D.D.U. Gorakhpur University, Gorakhpur (U.P.) – 273009, India; e-mail: sangeeta rahul@rediffmail.com Abstract: An acidic polygalacturonase (PG) secreted by Rhizopus oryzae MTCC-1987 in submerged fermentation condi- tion has been purified to electrophoretic homogeneity using ammonium sulphate fractionation and anion exchange chro- matography on diethylaminoethyl cellulose. The purified enzyme gave a single protein band in sodium dodecyl sulphate- polyacrylamide 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. Key words: polygalacturonase; submerged state fermentation; Rhizopus oryzae; enzymatic retting; Crotalaria juncea. Abbreviations: DEAE, diethylaminoethyl; DNSA, 3,5-dinitrosalicylic acid; EDTA, ethylenediaminetetraacetic acid; PG, polygalacturonase; PGA, polygalacturonic acid; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; TLC, thin layer chromatography. Introduction Pectin is a family of complex polysaccharides found in the middle lamella of primary cell walls of di- cotyledonous and non-graminaceous monocotyledonous plants. Pectin and hemicelluloses are responsible for the integrity and coherence of plant tissues as well as for the texture of vegetables and fruits (Voragen et al. 2009). These are mainly degraded by a group of enzymes re- ferred to as pectinases, which have been classified ac- cording to their mode of action and substrate prefer- ence into pectin esterases (EC 3.1.1.11), polygalactur- onases (PGs; EC 3.2.1.15), pectate lyases (EC 4.2.2.2) and pectin lyases (EC 4.2.2.10) (Yadav et al. 2009). Pectinases have been extensively reviewed and have po- tential applications in clarification of fruit juices, ret- ting of fibre, treatment of pectic waste water, coffee and tea leaf fermentation, oil extraction and virus pu- rifications (Jayani et al. 2005; Satyanarayana & Kumar 2005; Payasi et al. 2008; Pedrolli et al. 2009). PGs are pectinases that catalyze the hydrolysis of α-1,4-D-galacturonic acid linkages in smooth region of pectin (Parenicova et al. 2000; Torres et al. 2006) and are widely distributed among fungi, bacteria and yeasts along with higher plants and some parasitic ne- matodes (Sakai et al. 1993; Niture 2008). PG is one of the important members of pectin-degrading glycoside hydrolases of the family GH28. The sequence-structural features, specificities and evolutionary relationships of 115 PGs and related family GH28 sequences repre- senting bacterial, fungal, plant, insect groups have been attempted (Markovic & Janecek 2001). Depend- ing on their mode of hydrolysis of the substrate poly- mer, they are classified as endo-PGs (EC 3.2.1.15) and exo-PGs (EC 3.2.1.67). The endo-PGs cleave the α- 1,4-D-galacturonic linkages randomly, whereas exo-PGs cleave the linkage from the non-reducing end. The re- search work done on fungal PGs has been reviewed by Niture (2008). Most of the PGs studied so far belong to the endo-PGs group. PGs exhibit extensive multiplicity and variability as reported for those from Aspergillus species (Stratilova et al. 1993). Rhizopus oryzae also possesses a large PG gene family as elucidated from re- cently sequenced Rhizopus oryzae strain 99–880 with a total of 18 putative PG genes (Mertens et al. 2008). The expression and characterization of 15 PG genes comprising 12 endo-PGs and 3 exo-PGs of R. oryzae 99–880 in Pichia pastoris host along with biochemical characterization has recently been reported (Mertens & Bowman 2011). In search of PGs, the authors screened fungal strains belonging to different fungal genera. The fun- c 2012 Institute of Molecular Biology, Slovak Academy of Sciences