Purification of phenylalanine ammonia lyase (PAL) and peroxidase (POX) enzymes obtained from lucerne (Medicago sativa L. cv. Vertus) following inoculation with Verticillium albo-atrum
Keywords:
Ammonium sulphate precipitation, Verticillium, lucerne, PAL, POX
Abstract
Phenylalanine ammonia lyase (EC 4.3.1.24, PAL) and peroxidase (EC 1.11.1.7, POX) enzyme activities were determined in seedlings of lucerne (Medicago sativa L. cv. Vertus) after the inoculation of V. albo-atrum (V2, a weak pathogenic isolate to lucerne). PAL and POX activities of lucerne inoculated with V2 were found significantly higher than that of the control group after 48 h inoculation. Partial purification of enzymes with 0-50% (NH4)2SO4 precipitation in control groups resulted in 2.74 and 2.42 fold increases. On the other hand, 1.78 and 1.74 fold increases in PAL and POX activities, respectively were evident in V2-inoculated plants. This study showed that the purification of the enzymes with little effort enabled us to see the differences between the treatments clearly. This type of work could be suggested where the protein concentration of the sample is low or a time-scale experiment is to be carried out to see the enzyme changes or to characterize the pathogen-related proteins.
References
Agrios GN. 2002. Plant Pathology. Academic Press. 4th ed.
Altunkaya A, Gökmen V. 2011. Purification and characterization of polyphenol oxidase, peroxidase and lipoxygenase from freshly cut lettuce (L. sativa). Food Technol. Biotechnol., 49(2):249-256.
Boka K, Orban N. 2007. New aspect of H2O2 signaling. Plant Signal Behav., 2:498-500.
Bolwell GP, Bell J, Cramer J. Schuch W, Lamb CJ, and Dixon RA. 1985. L-phenylalanine ammonia-lyase from Phaseolus vulgaris. European J. of Biochem., 149:411-419.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 136:175-179.
Chutia J, Borah SP, Tanti B. 2012. Effect of drought stress on protein and proline metabolism in seven traditional rice (Oryza sativa Linn.) genotypes of Assam, India. Journal of Research in Biology, 2(3):206-214.
Civello PM, Arting GA, Chaves AR, Anan MC. 1995. Peroxidase from strawberry fruite by partial purification and determination of some properties. J. Agri. Food Chem., 43(10):2596-2601.
Das MK, Sharma RS, Mishra V. 2011. A novel cationic peroxidase (VanPrx) from a hemi-parasitic plant (Viscum angulatum) of western ghats (India): Purification, characterization and kinetic properties. J. Mol. Catal. B: Enzyme, 71:63-70.
Dikilitas M. 2003. Effect of salinity & its interactions with Verticillium albo-atrum on the disease development in tomato (Lycopersicon esculentum Mill.) and lucerne (Medicago sativa & M. media) Plants. Ph.D. Thesis, University Of Wales, Swansea, UK.
Dikilitas, M. 1997. A study of interactions between the phytopathogenic fungus Verticillium albo-atrum (Reinke & Berth) and tomato (Lycopersicon esculentum) and various cultivars of lucerne (Medicago sativa L. & M. media). MPhil thesis, University of Wales, Swansea, UK.
Dunn DC, Duncan LW, Romeo JT. 1998. Changes in arginine, PAL activity, and nematode behaviour in salinity-stressed citrus. Phytochemistry 49(2), 413-417.
Eldeen D, Radwan M, Fayez KA, Mahmoud SY, Lu G. 2010. Modifications of antioxidant activity and protein composition of bean leaf due to Bean yellow mosaic virus infection and salicylic acid treatments. Acta Physiol Plant., 32:891-904.
Hamid M, Rehman K. 2009. Potential applications of peroxidases. Food Chem., 115:1177-1186.
Hammerschmidt R. 1999. Induced disease resistance: how do induced plants stop pathogens? Physiol. Mol. Plant Pathol., 55:77-84.
Hu Y, Wu J, Luo P, Mo Y. 2012. Purification and partial characterization of peroxidase from lettuce stems. Afr. J. Biotechnol., 11(11):2752-2756.
Kim SS, Lee DJ. 2005. Purification and characterization of a cationic peroxidase Cs in Raphanus sativus. J. Plant. Physiol., 162:609-617.
Lavery CB, Macinnis MC, Macdonald MJ, Williams JB, Spencer CA, Burke AA, Irwin DJ, D'Cunha GB. 2010. Purification of peroxidase from Horseradish (Armoracia rusticana) roots. J. Agric. Food Chem., 58: 8471-8476.
Meriga B, Reddy BK, Rao KR, Reddy LA, Kavi Kishor PB. 2004. Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa). J. Plant Physiol., 161:63-68.
Mohamed SA, El-Badry MO, Drees EA, Fahmy AS. 2008. Properties of a Cationic Peroxidase from citrus jambhiri cv. Adalia. Appl. Biochem. Biotechnol., 150: 127-137.
Renugadevi R, Ayyappadas MP, Preethy PH, Savetha S. 2011. Isolation, screening and induction of mutation in strain for extra cellular lignin peroxidase producing bacteria from soil and its partial purification. Journal of Research in Biology, 4:312-318.
Silva ME, Franco TT. 2000. Purification of soybean peroxidase (Glycine max) by metal affinity partitioning in aqueous two-phase systems. J. Chromatogr. B 743: 287-294.
Tang M. 2001. Elicitor-Induced Defence Responses and Mechanisms of Signal Transduction in
Medicago sativa L. Ph.D. Thesis, University of Wales, Swansea.
Tsujimura H, Takaya M, Katano K, Matsumoto N, Park YS, Okabe M. 1994. Scaleup of peroxidase production by rthromyces ramosus based on analysis of fluid velocity distribution. J. Ferment. Bioeng., 77:650-654.
Zia MA, Kousar M, Ahmed I, Iqbal HMN, Abbas RZ. 2011. Comparative study of peroxidase purification from apple and orange seeds. Afr. J. Biotechnol., 10(33): 6300-6303.
Altunkaya A, Gökmen V. 2011. Purification and characterization of polyphenol oxidase, peroxidase and lipoxygenase from freshly cut lettuce (L. sativa). Food Technol. Biotechnol., 49(2):249-256.
Boka K, Orban N. 2007. New aspect of H2O2 signaling. Plant Signal Behav., 2:498-500.
Bolwell GP, Bell J, Cramer J. Schuch W, Lamb CJ, and Dixon RA. 1985. L-phenylalanine ammonia-lyase from Phaseolus vulgaris. European J. of Biochem., 149:411-419.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 136:175-179.
Chutia J, Borah SP, Tanti B. 2012. Effect of drought stress on protein and proline metabolism in seven traditional rice (Oryza sativa Linn.) genotypes of Assam, India. Journal of Research in Biology, 2(3):206-214.
Civello PM, Arting GA, Chaves AR, Anan MC. 1995. Peroxidase from strawberry fruite by partial purification and determination of some properties. J. Agri. Food Chem., 43(10):2596-2601.
Das MK, Sharma RS, Mishra V. 2011. A novel cationic peroxidase (VanPrx) from a hemi-parasitic plant (Viscum angulatum) of western ghats (India): Purification, characterization and kinetic properties. J. Mol. Catal. B: Enzyme, 71:63-70.
Dikilitas M. 2003. Effect of salinity & its interactions with Verticillium albo-atrum on the disease development in tomato (Lycopersicon esculentum Mill.) and lucerne (Medicago sativa & M. media) Plants. Ph.D. Thesis, University Of Wales, Swansea, UK.
Dikilitas, M. 1997. A study of interactions between the phytopathogenic fungus Verticillium albo-atrum (Reinke & Berth) and tomato (Lycopersicon esculentum) and various cultivars of lucerne (Medicago sativa L. & M. media). MPhil thesis, University of Wales, Swansea, UK.
Dunn DC, Duncan LW, Romeo JT. 1998. Changes in arginine, PAL activity, and nematode behaviour in salinity-stressed citrus. Phytochemistry 49(2), 413-417.
Eldeen D, Radwan M, Fayez KA, Mahmoud SY, Lu G. 2010. Modifications of antioxidant activity and protein composition of bean leaf due to Bean yellow mosaic virus infection and salicylic acid treatments. Acta Physiol Plant., 32:891-904.
Hamid M, Rehman K. 2009. Potential applications of peroxidases. Food Chem., 115:1177-1186.
Hammerschmidt R. 1999. Induced disease resistance: how do induced plants stop pathogens? Physiol. Mol. Plant Pathol., 55:77-84.
Hu Y, Wu J, Luo P, Mo Y. 2012. Purification and partial characterization of peroxidase from lettuce stems. Afr. J. Biotechnol., 11(11):2752-2756.
Kim SS, Lee DJ. 2005. Purification and characterization of a cationic peroxidase Cs in Raphanus sativus. J. Plant. Physiol., 162:609-617.
Lavery CB, Macinnis MC, Macdonald MJ, Williams JB, Spencer CA, Burke AA, Irwin DJ, D'Cunha GB. 2010. Purification of peroxidase from Horseradish (Armoracia rusticana) roots. J. Agric. Food Chem., 58: 8471-8476.
Meriga B, Reddy BK, Rao KR, Reddy LA, Kavi Kishor PB. 2004. Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa). J. Plant Physiol., 161:63-68.
Mohamed SA, El-Badry MO, Drees EA, Fahmy AS. 2008. Properties of a Cationic Peroxidase from citrus jambhiri cv. Adalia. Appl. Biochem. Biotechnol., 150: 127-137.
Renugadevi R, Ayyappadas MP, Preethy PH, Savetha S. 2011. Isolation, screening and induction of mutation in strain for extra cellular lignin peroxidase producing bacteria from soil and its partial purification. Journal of Research in Biology, 4:312-318.
Silva ME, Franco TT. 2000. Purification of soybean peroxidase (Glycine max) by metal affinity partitioning in aqueous two-phase systems. J. Chromatogr. B 743: 287-294.
Tang M. 2001. Elicitor-Induced Defence Responses and Mechanisms of Signal Transduction in
Medicago sativa L. Ph.D. Thesis, University of Wales, Swansea.
Tsujimura H, Takaya M, Katano K, Matsumoto N, Park YS, Okabe M. 1994. Scaleup of peroxidase production by rthromyces ramosus based on analysis of fluid velocity distribution. J. Ferment. Bioeng., 77:650-654.
Zia MA, Kousar M, Ahmed I, Iqbal HMN, Abbas RZ. 2011. Comparative study of peroxidase purification from apple and orange seeds. Afr. J. Biotechnol., 10(33): 6300-6303.
Published
2012-06-01
How to Cite
Dikilitas, M. (2012). Purification of phenylalanine ammonia lyase (PAL) and peroxidase (POX) enzymes obtained from lucerne (Medicago sativa L. cv. Vertus) following inoculation with Verticillium albo-atrum. Journal of Research in Biology, 2(4), 355-361. Retrieved from https://ojs.jresearchbiology.com/ojs1/index.php/jrb/article/view/224
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