Skip to content
BY-NC-ND 3.0 license Open Access Published by De Gruyter October 19, 2012

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

  • Sangeeta Yadav EMAIL logo , Gautam Anand , Amit Dubey and Dinesh Yadav
From the journal Biologia

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 K m and k cat values of the PG were 2.7 mg/mL and 2.23 × 103 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+, Hg2+ and KMnO4, while it was stimulated to some extent by Co2+. The purified PG exhibited retting of Crotalaria juncea fibre in absence of ethylenediaminetetraacetic acid.

[1] Barense R.I., dos S.C. Chellegatti M.A., Fonseca M.J. & Said S. 2001. Partial purification and characterization of exopolygalacturonase II and III of Penicillium frequentans. Braz. J. Microbiol. 32: 327–330. http://dx.doi.org/10.1590/S1517-8382200100040001410.1590/S1517-83822001000400014Search in Google Scholar

[2] Devi N.A. & AppuRao A.G. 1996. Fractionation, purification, and preliminary characterization of polygalacturonases produced by Aspergillus carbonarius. Enzyme Microb. Technol. 18: 59–65. http://dx.doi.org/10.1016/0141-0229(96)00055-510.1016/0141-0229(96)00055-5Search in Google Scholar

[3] Engel P.C. 1977. Enzyme Kinetics. A Steady State Approach. Chapman and Hall, London. Search in Google Scholar

[4] Foda M.S., Rizk I.R.S., Gibriel A.Y & Basha A.Y. 1984. Biochemical properties of polygalacturonase, produced by Aspergillus aculeatus and Mucor pusillus. Zentralblatt für Mikrobiologie 139: 463–469. Search in Google Scholar

[5] Henriksson G., Akin D.E., Rigsby L.L., Patel N. & Eriksson K.E.L. 1997. Influence of chelating agents and mechanical pretreatment on enzymatic retting of flax. Tex. Res. J. 67: 829–836. 10.1177/004051759706701107Search in Google Scholar

[6] Henriksson G., Akin D.E., Slomczynski D. & Eriksson K.E.L. 1999. Production of highly efficient enzymes for flax retting by Rhizomucor pusillus. J. Biotechnol. 68: 115–123. http://dx.doi.org/10.1016/S0168-1656(98)00192-810.1016/S0168-1656(98)00192-8Search in Google Scholar

[7] Jacob N., Asha Poorna C. & Prema P. 2008. Purification and partial characterization of polygalacturonase from Streptomyces lydicus. Bioresour. Technol. 99: 6697–6701. http://dx.doi.org/10.1016/j.biortech.2007.10.00210.1016/j.biortech.2007.10.002Search in Google Scholar PubMed

[8] Jayani S.R., Shivalika S. & Gupta R. 2005. Microbial pectinolytic enzymes: a review. Process Biochem. 40: 2931–2944. http://dx.doi.org/10.1016/j.procbio.2005.03.02610.1016/j.procbio.2005.03.026Search in Google Scholar

[9] Kaur G., Kumar S. & Satyanarayana T. 2004. Production, characterization and application of a thermostable polygalacturonase of a thermophilic mould Sporotrichum thermophile Apinis. Bioresour. Technol. 94: 239–243. http://dx.doi.org/10.1016/j.biortech.2003.05.00310.1016/j.biortech.2003.05.003Search in Google Scholar PubMed

[10] Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. http://dx.doi.org/10.1038/227680a010.1038/227680a0Search in Google Scholar PubMed

[11] Manjon A., Iborra J.L., Romero C. & Canovas M. 1992. Properties of pectinesterase and endo-d-polygalacturonase coimmobilized in a porous glass support. Appl. Biochem. Biotechnol. 37: 19–31. http://dx.doi.org/10.1007/BF0278885410.1007/BF02788854Search in Google Scholar

[12] Markovic O. & Janecek S. 2001. Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution. Protein Eng. 14: 615–631. http://dx.doi.org/10.1093/protein/14.9.61510.1093/protein/14.9.615Search in Google Scholar PubMed

[13] Martin N., De Souza S.R., Da Silva R. & Gomes E. 2004. Pectinase production by fungal strains in solid-state fermentation using agro-industrial bioproduct. Braz. Arch. Biol. Technol. 47: 813–819. http://dx.doi.org/10.1590/S1516-8913200400050001810.1590/S1516-89132004000500018Search in Google Scholar

[14] Mertens J.A., Burdick R.C. & Rooney A.P. 2008. Identification, biochemical characterisation and evolution of the Rhizopus oryzae 99–880 polygalacturonase family. Fungal Genet. Biol. 45: 1616–1624. http://dx.doi.org/10.1016/j.fgb.2008.09.00910.1016/j.fgb.2008.09.009Search in Google Scholar PubMed

[15] Mertens J.A & Bowman M.J. 2011. Expression and characterization of fifteen Rhizopus oryzae 99–880 polygalacturonase enzymes in Pichia pastoris. Curr. Microbiol. 62: 1173–1178. http://dx.doi.org/10.1007/s00284-010-9842-810.1007/s00284-010-9842-8Search in Google Scholar PubMed

[16] Miller G.L. 1959. Use of dinitrosalicyclic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426–428. http://dx.doi.org/10.1021/ac60147a03010.1021/ac60147a030Search in Google Scholar

[17] Molina S.M.G., Pelissari F.A. & Vitorello C.B.M. 2001. Screening and genetic improvement of pectinolytic fungi for degumming of textile fibres. Braz. J. Microbiol. 32: 320–326. http://dx.doi.org/10.1590/S1517-8382200100040001310.1590/S1517-83822001000400013Search in Google Scholar

[18] Niture S.K. 2008. Comparative biochemical and structural characterizations of fungal polygalacturonases. Biologia 63: 1–19. http://dx.doi.org/10.2478/s11756-008-0018-y10.2478/s11756-008-0018-ySearch in Google Scholar

[19] Ortega N., De Diego S., Rodriguez-Nogales J.M., Perez-Mateos M. & Busto M.D. 2004. Kinetic behaviour and thermal inactivation of pectin lyase used in food processing. J. Food Sci. Technol. 39: 631–639. http://dx.doi.org/10.1111/j.1365-2621.2004.00822.x10.1111/j.1365-2621.2004.00822.xSearch in Google Scholar

[20] Parenicova L., Benen J.A., Kester H.C. & Visser J. 1998. pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. Eur. J. Biochem. 251: 72–80. http://dx.doi.org/10.1046/j.1432-1327.1998.2510072.x10.1046/j.1432-1327.1998.2510072.xSearch in Google Scholar PubMed

[21] Parenicova L., Benen J.A., Kester H.C. & Visser J. 2000. pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger. Biochem. J. 345: 637–644. http://dx.doi.org/10.1042/0264-6021:345063710.1042/bj3450637Search in Google Scholar

[22] Payasi A., Sanwal R. & Sanwal G.G. 2008. Microbial pectate lyases: characterization and enzymological properties. World J. Microbiol. Biotechnol. 25: 1–14. http://dx.doi.org/10.1007/s11274-008-9870-810.1007/s11274-008-9870-8Search in Google Scholar

[23] Pedrolli D.B., Monteiro A.C., Gomes E. & Carmona E.C. 2009. Pectin and pectinases: production, characterization and industrial application of microbial pectinolytic enzymes. Open Biotechnol. J. 3: 9–18. http://dx.doi.org/10.2174/187407070090301000910.2174/1874070700903010009Search in Google Scholar

[24] Rao M.N., Kembhavi A.A. & Pant A. 1996. Implication of tryptophan and histidine in the active site of endo-polygalacturonase from Aspergillus ustus: elucidation of the reaction mechanism. Biochim. Biophys. Acta. 1296: 167–173. http://dx.doi.org/10.1016/0167-4838(96)00067-210.1016/0167-4838(96)00067-2Search in Google Scholar

[25] Rihouey C., Jauneau A., Cabin-Flaman A., Demarty M., Lefevre F. & Morvan C. 1995. Calcium and acidic pectin distribution in flax cell walls: evidence for different kinds of linkages in the cell junction and middle lamella of the cortical parenchyma of flax hypocotyl. Plant Physiol. Biochem. 33: 497–508. Search in Google Scholar

[26] Saito K., Takakuwa N. & Oda Y. 2004. Purification of the extracellular pectinolytic enzyme from the fungus Rhizopus oryzae NBRC 4707. Microbiol. Res. 159: 83–86. http://dx.doi.org/10.1016/j.micres.2004.01.00110.1016/j.micres.2004.01.001Search in Google Scholar

[27] Sakai T., Sakamoto T., Hallaert J. & Vandamme E.J. 1993. Pectin, pectinase and protopectinase: production, properties and applications. Adv. Appl. Microbiol. 39: 213–294. http://dx.doi.org/10.1016/S0065-2164(08)70597-510.1016/S0065-2164(08)70597-5Search in Google Scholar

[28] Sakamoto T., Bonnin E., Quemener B. & Thibault J.F. 2002. Purification and characterisation of two exo-polygalacturonases from Aspergillus niger able to degrade xylogalacturonan and acetylated homogalacturonan. Biochim. Biophys. Acta. 1572: 10–18. http://dx.doi.org/10.1016/S0304-4165(02)00277-510.1016/S0304-4165(02)00277-5Search in Google Scholar

[29] Satyanarayana N.G. & Kumar D.S. 2005. Microbial pectic transeliminases. Biotechnol. Lett. 27: 451–458. http://dx.doi.org/10.1007/s10529-005-2197-810.1007/s10529-005-2197-8Search in Google Scholar PubMed

[30] Semenova M.V., Grishutin S.G., Gusakov A.V., Okunev O.N. & Sinitsyn A.P. 2003. Isolation and properties of pectinases from the fungus Aspergillus japonicus. Biochemistry (Moscow) 68: 559–569. http://dx.doi.org/10.1023/A:102395972706710.1023/A:1023959727067Search in Google Scholar

[31] Stratilova E., Markovic O., Skrovimova D., Rexova-Benkova L. & Jornvall H. 1993. Pectinase Aspergillus sp. polygalacturonase: multiplicity, divergence and structural patterns linking fungal bacterial and plant polygalacturonases. J. Protein Chem. 12: 15–22. http://dx.doi.org/10.1007/BF0102490910.1007/BF01024909Search in Google Scholar PubMed

[32] Tari C., Dogan N. & Gogus N. 2008. Biochemical and thermal characterization of crude exo-polygalacturonase produced by Aspergillus sojae. Food Chem. 111: 824–829. http://dx.doi.org/10.1016/j.foodchem.2008.04.05610.1016/j.foodchem.2008.04.056Search in Google Scholar

[33] Thakur A., Pahwa R., Singh S. & Gupta R. 2010. Production, purification, and characterization of polygalacturonase from Mucor circinelloides ITCC 6025. Enzyme Res. Article ID 170549. Search in Google Scholar

[34] Torres E.F., Sepulveda T.V. & Gonzalez G.V. 2006. Production of hydrolytic depolymerising pectinases. Food Technol. Biotechnol 44: 221–227. Search in Google Scholar

[35] Vazquez C., Patino B. & Martinez M.J. 1993. Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici. FEMS Microbiol. Lett. 110:191–196. http://dx.doi.org/10.1111/j.1574-6968.1993.tb06319.x10.1111/j.1574-6968.1993.tb06319.xSearch in Google Scholar

[36] Voragen A.G.J., Conen G.J., Verhof R.P. & Schols H.A. 2009. Pectin, a versatile polysaccharide present in plant cell walls. Struct. Chem. 20: 263–275. http://dx.doi.org/10.1007/s11224-009-9442-z10.1007/s11224-009-9442-zSearch in Google Scholar

[37] Yadav S., Yadav P.K., Yadav D. & Yadav K.D.S. 2009. Pectin lyase: a review. Process Biochem. 44: 1–10. http://dx.doi.org/10.1016/j.procbio.2008.09.01210.1016/j.procbio.2008.09.012Search in Google Scholar

[38] Zhang J., Henriksson H., Szabo I.J., Henriksson G. & Johansson G. 2005. The active component in the flax retting system of the zygomycetes Rhizopus oryzae sb. is a family 28 polygalacturonase. J. Ind. Microbiol. Biotechnol. 32: 431–438. http://dx.doi.org/10.1007/s10295-005-0014-y10.1007/s10295-005-0014-ySearch in Google Scholar PubMed

Published Online: 2012-10-19
Published in Print: 2012-12-1

© 2012 Slovak Academy of Sciences

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Downloaded on 28.3.2024 from https://www.degruyter.com/document/doi/10.2478/s11756-012-0122-x/html
Scroll to top button