Recombinant Expression and Characterization of Lemon (Citrus limon) Peroxidase

Author(s): Veda P. Pandey, Apoorvi Tyagi, Shagoofa Ali, Kusum Yadav, Anurag Yadav, Ajit K. Shasany, Upendra N. Dwivedi*

Journal Name: Protein & Peptide Letters

Volume 28 , Issue 4 , 2021

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background: Class III plant peroxidases play important role in a number of physiological processes in plants such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable.

Objective: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application.

Methods: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols.

Results: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously in E. coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+, Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and shown the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms.

Conclusion: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

Keywords: Functional annotation, in-vitro folding, in-silico analyses, molecular modeling, phenylpropanoid pathway, recombinant expression.

Welinder, K.G. Superfamily of plant, fungal and bacterial peroxidases. Curr. Opin. Struct. Biol., 1992, 2, 388-393.
Pandey, V.P.; Awasthi, M.; Singh, S.; Tiwari, S.; Dwivedi, U.N. A comprehensive review on function and application of plant peroxidases. Biochem. Anal. Biochem., 2017, 6, 308-323.
Husain, Q. Peroxidase mediated decolorization and remediation of wastewater containing industrial dyes: a review. Rev. Environ. Sci. Biotechnol., 2010, 9, 117-140.
Regalado Garc’ıa-Almend’arez, C.E.; Duarte-V’azquez, M.A. Biotechnological applications of peroxidases. Phytochem. Rev., 2004, 3, 243-256.
Pandey, V.P.; Dwivedi, U.N. A ripening associated peroxidase from papaya having a role in defense and lignification: heterologous expression and in-silico and in-vitro experimental validation. Gene, 2015, 555(2), 438-447.
[] [PMID: 25447898]
Violante-Mota, F.; Tellechea, E.; Moran, J.F.; Sarath, G.; Arredondo-Peter, R. Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants. Phytochemistry, 2010, 71(1), 21-26.
[] [PMID: 19833360]
Fattahian, Y.; Riahi-Madvar, A.; Mirzaee, R.; Torkzadeh-Mahani, M.; Asadikaram, G. Heterologous expression, purification and characterization of a peroxidase isolated from Lepidium draba. Protein J., 2017, 36(6), 461-471.
[] [PMID: 28975545]
Rodríguez-Cabrera, N.A.; Regalado, C.; García-Almendárez, B.E. Cloning, heterologous expression and properties of a recombinant active turnip peroxidase. J. Agric. Food Chem., 2011, 59(13), 7120-7126.
[] [PMID: 21591783]
Pham, T.M.; Kim, S.J.; Song, B.K.; Kim, Y.H. Optimized refolding and characterization of S-peroxidase (CWPO_C of Populus alba) expressed in E. coli. Protein Expr. Purif., 2011, 80(2), 268-273.
[] [PMID: 21856428]
Bae, E.K.; Lee, H.; Lee, J.S.; Noh, E.W.; Jo, J. Molecular cloning of a peroxidase gene from poplar and its expression in response to stress. Tree Physiol., 2006, 26(11), 1405-1412.
[] [PMID: 16877325]
Spadiut, O.; Rossetti, L.; Dietzsch, C.; Herwig, C. Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy. Protein Expr. Purif., 2012, 86(2), 89-97.
[] [PMID: 23026679]
Krainer, F.W.; Pletzenauer, R.; Rossetti, L.; Herwig, C.; Glieder, A.; Spadiut, O. Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris. Protein Expr. Purif., 2014, 95, 104-112.
[] [PMID: 24342173]
Green, M.R.; Sambrook, J. Molecular Cloning: A Laboratory Manual, 4th Ed; Cold Spring Harbor Laboratory: America, 2012.
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 1976, 72, 248-254.
[] [PMID: 942051]
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680-685.
[] [PMID: 5432063]
Pandey, V.P.; Dwivedi, U.N. Purification and characterization of peroxidase from Leucaena leucocephala, a tree legume. J. Mol. Catal., B Enzym., 2011, 68, 168-173.
Davis, B.J. Disc Electrophoresis II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci., 1964, 121, 404-427.
[] [PMID: 14240539]
Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res., 2001, 29(9)e45
[] [PMID: 11328886]
Asad, S.; Dabirmanesh, B.; Ghaemi, N.; Etezad, S.M.; Khajeh, K. Studies on the refolding process of recombinant horseradish peroxidase. Mol. Biotechnol., 2013, 54(2), 484-492.
[] [PMID: 22872497]
Gabaldón, C.; López-Serrano, M.; Pedreño, M.A.; Barceló, A.R. Cloning and molecular characterization of the basic peroxidase isoenzyme from Zinnia elegans, an enzyme involved in lignin biosynthesis. Plant Physiol., 2005, 139(3), 1138-1154.
[] [PMID: 16258008]
Liu, C.; Zheng, K.; Xu, Y.; Stephen, L.T.; Wang, J.; Zhao, H.; Yue, T.; Nian, R.; Zhang, H.; Xian, M.; Liu, H. Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3). Prep. Biochem. Biotechnol., 2017, 47(8), 768-775.
[] [PMID: 28644760]
Pandey, V.P.; Rani, J.; Jaiswal, N.; Singh, S.; Awasthi, M.; Shasany, A.K.; Tiwari, S.; Dwivedi, U.N. Chitosan immobilized novel peroxidase from Azadirachta indica: Characterization and application. Int. J. Biol. Macromol., 2017, 104(Pt B), 1713-1720.
[] [PMID: 28215563]
Fortea, M.I.; Pellicer, J.A.; Serrano-Martínez, A.; López-Miranda, S.; Lucas-Abellán, C.; Núñez-Delicado, E. Red cabbage (Brassica oleracea) as a new source of high-thermostable peroxidase. J. Agric. Food Chem., 2012, 60(42), 10641-10648.
[] [PMID: 23013444]
Hu, Y.; Wu, J.; Luo, P.; Mo, Y. Purification and partial characterization of peroxidase from lettuce stems. Afr. J. Biotechnol., 2012, 11, 2752-2756.
Mall, R.; Naik, G.; Mina, U.; Mishra, S.K. Purification and characterization of a thermostable soluble peroxidase from Citrus medica leaf. Prep. Biochem. Biotechnol., 2013, 43(2), 137-151.
[] [PMID: 23302102]
Mohamed, S.A.; El-Badry, M.O.; Drees, E.A.; Fahmy, A.S. Properties of a cationic peroxidase from Citrus jambhiri cv. Adalia. Appl. Biochem. Biotechnol., 2008, 150(2), 127-137.
[] [PMID: 18633734]
Adams, J.B. Regeneration and the kinetics of peroxidase inactivation. Food Chem., 1997, 60, 201-206.
Converso, D.A.; Fernández, M.E. Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control. Arch. Biochem. Biophys., 1996, 333(1), 59-65.
[] [PMID: 8806754]
Sasaki, K.; Yuichi, O.; Hiraga, S.; Gotoh, Y.; Seo, S.; Mitsuhara, I.; Ito, H.; Matsui, H.; Ohashi, Y. Characterization of two rice peroxidase promoters that respond to blast fungus-infection. Mol. Genet. Genomics, 2007, 278(6), 709-722.
[] [PMID: 17805575]
Lavania, M.; Chauhan, P.S.; Chauhan, S.V.; Singh, H.B.; Nautiyal, C.S. Induction of plant defense enzymes and phenolics by treatment with plant growth-promoting rhizobacteria Serratia marcescens NBRI1213. Curr. Microbiol., 2006, 52(5), 363-368.
[] [PMID: 16586018]
Thordal-Christensen, H.; Brandt, J.; Cho, B.H.; Rasmussen, S.K.; Gregersen, P.L.; Smedegaard-Petersen, V.; Collinge, D.B. cDNA cloning and characterization of two barley transcripts induced differentially by powdery mildew fungus Erysiphie graminis. Physiol. Mol. Plant Pathol., 1992, 40, 395-409.
Singh, D.P.; Singh, V.; Gupta, V.K.; Shukla, R.; Prabha, R.; Sarma, B.K.; Patel, J.S. Microbial inoculation in rice regulates antioxidative reactions and defense related genes to mitigate drought stress. Sci. Rep., 2020, 10(1), 4818.
[] [PMID: 32179779]
Ogawa, S.; Shiro, Y.; Morishima, I. Calcium binding by horseradish peroxidase C and the heme environmental structure. Biochem. Biophys. Res. Commun., 1979, 90(2), 674-678.
[] [PMID: 508331]
Almargo, L.G.O.; Mezros, L.V.; Bru, S.; Belchi-navarro, R. Ros Barceló. A.; Pedreno, M.A. Class III peroxidases in plant defense reactions. J. Exp. Bot., 2009, 60, 377-390.
[] [PMID: 19073963]
Gajhede, M. Plant peroxidases: substrate complexes with mechanistic implications. Biochem. Soc. Trans., 2001, 29(Pt 2), 91-98.
[] [PMID: 11356134]
Itakura, H.; Oda, Y.; Fukuyama, K. Binding mode of benzhydroxamic acid to Arthromyces ramosus peroxidase shown by X-ray crystallographic analysis of the complex at 1.6 A resolution. FEBS Lett., 1997, 412(1), 107-110.
[] [PMID: 9257700]
Henriksen, A.; Schuller, D.J.; Meno, K.; Welinder, K.G.; Smith, A.T.; Gajhede, M. Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography. Biochemistry, 1998, 37(22), 8054-8060.
[] [PMID: 9609699]
Nokthai, P.; Lee, V.S.; Shank, L. Molecular modeling of peroxidase and polyphenol oxidase: substrate specificity and active site comparison. Int. J. Mol. Sci., 2010, 11(9), 3266-3276.
[] [PMID: 20957092]
Tatoli, S.; Zazza, C.; Sanna, N.; Palma, A.; Aschi, M. The role of arginine 38 in horseradish peroxidase enzyme revisited: a computational investigation. Biophys. Chem., 2009, 141(1), 87-93.
[] [PMID: 19178992]
Rodriguez-Lopez, J.N.; Smith, A.T.; Thorneley, R.N. Role of arginine 38 in horseradish peroxidase. A critical residue for substrate binding and catalysis. J. Biol. Chem., 1996, 271(8), 4023-4030.
[] [PMID: 8626735]
Passardi, F.; Cosio, C.; Penel, C.; Dunand, C. Peroxidases have more functions than a Swiss army knife. Plant Cell Rep., 2005, 24(5), 255-265.
[] [PMID: 15856234]
Cosio, C.; Dunand, C. Specific functions of individual class III peroxidase genes. J. Exp. Bot., 2009, 60(2), 391-408.
[] [PMID: 19088338]
Christensen, J.H.; Bauw, G.; Welinder, K.G.; Van Montagu, M.; Boerjan, W. Purification and characterization of peroxidases correlated with lignification in poplar xylem. Plant Physiol., 1998, 118(1), 125-135.
[] [PMID: 9733532]
Lin, Y.; Li, W.; Zhang, Y.; Xia, C.; Liu, Y.; Wang, C.; Xu, R.; Zhang, L.; Zhang, L. Identification of genes/proteins related to submergence tolerance by transcriptome and proteome analyses in soybean. Sci. Rep., 2019, 9(1), 14688.
[] [PMID: 31604973]
Moural, T.W.; Lewis, K.M.; Barnaba, C.; Zhu, F.; Palmer, N.A.; Sarath, G.; Scully, E.D.; Jones, J.P.; Sattler, S.E.; Kang, C. Characterization of class III peroxidases from switchgrass. Plant Physiol., 2017, 173(1), 417-433.
[] [PMID: 27879392]

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2021
Published on: 19 May, 2021
Page: [469 - 479]
Pages: 11
DOI: 10.2174/0929866527666200925114054
Price: $65

Article Metrics

PDF: 28