Guaiakol Peroksidazın Soğan Köklerinden Afinite Kromatografisi ile Saflaştırılması

Peroksidazlar, hidrojen peroksit varlığında çeşitli organik substratların oksidasyonunu katalizleyen ve yapılarında hem grubu bulunduran enzimlerdir. Özellikle bitki peroksidazları, endüstride, klinik tanıda, biyosensör yapımında ve organik sentez reaksiyonlarında sıklıkla kullanılır. Ticari değerleri nedeniyle bu enzimlerin farklı kaynaklarda tanımlanması ve saflaştırılması büyük öneme sahiptir. Bu çalışmada peroksidaz enzimi ilk kez aminobenzohydrazide tabanlı afinite kromatografi tekniği kullanılarak soğan köklerinden 37.7 verimle 750 kat saflaştırıldı. Saflaştırılan enzimin moleküler ağırlığını belirlemek için SDS-PAGE yapıldı ve 51.2 kDa’da tek bant gözlendi. Ayrıca enzimin guaiakol, ABTS ve pirogallol substratları için KM değerleri sırasıyla 3.44, 0.46 ve 21.27 mM olarak olarak hesaplandı.

Anahtar Kelimeler:

Soğan (Allium cepa) kökü, peroksidaz, afinite kromatografisi, saflaştırma

Separation of Guaiacol Peroxidase from Onion Roots with Affinity Chromatography

Peroxidases are enzymes that catalyze the oxidation of various organic substrates in the presence of hydrogen peroxide and contain heme group in their structure. In particular, plant peroxidases are frequently used in industry, clinical diagnosis, biosensor construction and organic synthesis reactions. Due to their commercial value, the identification and purification of these enzymes in different sources is of great importance. In this study, peroxidase enzyme was purified for the first time by using aminobenzohydrazide-based affinity chromatography technique from onion roots 750-fold with a yield of 37.7. SDS-PAGE was performed to determine the molecular weight of the purified enzyme and a single band was observed at 51.2 kDa. In addition, KM values of the enzyme for guaiacol, ABTS and pyrogallol substrates were calculated as 3.44, 0.46 and 21.27 mM, respectively.

Keywords:

Onion (Allium cepa) root, peroxidase, affinity chromatography, purification,

PDF

___

Adam W, Lazarus M, Saha-Möller CR, Weichold O, Hoch U, Häring D, Schreier P, 1999. Biotransformations with peroxidases, Biotransformations, Springer, pp. 73-108, Berlin-Germany.
Agostini E, Hernández‐Ruiz J, Arnao MB, Milrad SR, Tigier HA, Acosta M, 2002. A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy‐root cultures: inactivation by hydrogen peroxide and application in diagnostic kits. Biotechnology and applied biochemistry, 35(1): 1-7.
Bach CE, Warnock DD, Van Horn DJ, Weintraub MN, Sinsabaugh RL, Allison SD, German DP, 2013. Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: effect of assay conditions and soil type. Soil Biology and Biochemistry, 67: 183-191.
Bhunia A, Durani S, Wangikar, PP, 2001. Horseradish peroxidase catalyzed degradation of industrially important dyes. Biotechnology and Bioengineering, 72(5): 562-567.
Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 72(1-2): 248-254.
Chivukula M, Spadaro JT, Renganathan V, 1995. Lignin peroxidase-catalyzed oxidation of sulfonated azo dyes generates novel sulfophenyl hydroperoxides. Biochemistry, 34(23): 7765-7772.
El Agha A, Makris DP, Kefalas P, 2008. Hydrocaffeic acid oxidation by a peroxidase homogenate from onion solid wastes. European Food Research and Technology, 227(5): 1379-1386.
Gu TY, Hsu KH, Syu MJ, 2003. Scale-up of affinity chromatography for purification of enzymes and other proteins. Enzyme Microb Tech, 33: 430-437.
Heller A, Vreeke MS, 1997. Soybean peroxidase electrochemical sensor. In: Google Patents.
Hu Y, Wu J, Luo P, Mo Y, 2012. Purification and partial characterization of peroxidase from lettuce stems. African Journal of Biotechnology, 11(11): 2752-2756.
Jacob BM, Antony KE, Sreekumar B, Haridas M, 2000. Thiocyanate mediated antifungal and antibacterial property of goat milk lactoperoxidase. Life sciences, 66(25): 2433-2439.
Jia J, Wang B, Wu A, Cheng G, Li Z, Dong S, 2002. A method to construct a third-generation horseradish peroxidase biosensor: self-assembling gold nanoparticles to three-dimensional sol-gel network. Analytical Chemistry, 74(9): 2217-2223.
Kwak SS, Kim SK, Park IH, Liu JR, 1996. Enhancement of peroxidase activity by stress-related chemicals in sweet potato. Phytochemistry, 43(3): 565-568.
Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature, 227(5259): 680.
Lavery CB, MacInnis MC, MacDonald MJ, Williams JB, Spencer CA, Burke AA, D’Cunha GB, 2010. Purification of peroxidase from horseradish (Armoracia rusticana) roots. Journal of agricultural and food chemistry, 58(15): 8471-8476.
Liu J, Ye L, Weiping Y, 1999. Copolymerization of lignin with cresol catalysed by peroxidase in reversed micellar systems. Electronic Journal of Biotechnology, 2(2): 7-8.
Mall R, Naik G, Mina U, Mishra SK, 2013. Purification and characterization of a thermostable soluble peroxidase from Citrus medica leaf. Preparative Biochemistry and Biotechnology, 43(2): 137-151.
Merril CR 1990. Gel-staining techniques. Methods in enzymology Vol. 182, pp. 477-488, Elsevier.
Mohamed SA, El-Badry MO, Drees EA, Fahmy AS, 2008. Properties of a cationic peroxidase from Citrus jambhiri cv. Adalia. Applied biochemistry and biotechnology, 150(2): 127-137.
Moussouni S, Detsi A, Majdalani M, Makris DP, Kefalas P, 2010. Crude peroxidase from onion solid waste as a tool for organic synthesis. Part I: Cyclization of 2′, 3, 4, 4′, 6′-pentahydroxy-chalcone into aureusidin. Tetrahedron Letters, 51(31): 4076-4078.
Osman A, Makris D, 2011. Oxidation of morin (2', 3, 4', 5, 7-Pentahydroxylavone) with a peroxidase homogenate from onion. International Food Research Journal, 18(3).
Oztekin A, Almaz Z, Gerni S, Erel D, Kocak SM, Sengül ME, Ozdemir H, 2019. Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique. Journal of Chromatography B, 1114: 86-92.
Oztekin A, 2019. Determination of some flavonoid derivatives inhibitory effect on bovine milk lactoperoxidase enzyme, Turkish journal of science, 4(1): 15-21.
Pandey VP, Awasthi M, Singh S, Tiwari S, Dwivedi UN, 2017. A comprehensive review on function and application of plant peroxidases. Biochemistry and Analytical Biochemistry, 6(1): 308.
Pandey VP, Singh S, Singh R, Dwivedi UN, 2012. Purification and characterization of peroxidase from papaya (Carica papaya) fruit. Applied biochemistry and biotechnology, 167(2): 367-376.
Passardi F, Cosio C, Penel C, Dunand C, 2005. Peroxidases have more functions than a Swiss army knife. Plant cell reports, 24(5): 255-265.
Regalado C, García-Almendárez BE, Duarte-Vázquez MA, 2004. Biotechnological applications of peroxidases. Phytochemistry Reviews, 3(1-2): 243-256.
Sakharov IY, 2001. Long-term chemiluminescent signal is produced in the course of luminol peroxidation catalyzed by peroxidase isolated from leaves of African oil palm tree. Biochemistry (Moscow), 66(5): 515-519.
Soda I, Hasegawa T, Suzuki T, Ogura N, 1991. Purification and some properties of peroxidase from kiwifruit. Agricultural and biological chemistry, 55(6): 1677-1678.
Takahama U, 2004. Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: physiological significance of the oxidation reactions. Phytochemistry Reviews, 3(1-2): 207-219.
Tatsumi K, Wada S, Ichikawa H, 1996. Removal of chlorophenols from wastewater by immobilized horseradish peroxidase. Biotechnology and Bioengineering, 51(1): 126-130.
Thongsook T, Barrett DM, 2005. Heat inactivation and reactivation of broccoli peroxidase. Journal of agricultural and food chemistry, 53(8): 3215-3222.
Wilchek M, Chaiken I, 2000. An overview of affinity chromatography. Affinity Chromatography, pp. 1-6: Springer.
Zilbeyaz K, Kilic H, Sisecioglu M, Ozdemir H, Güngör AA, 2012. Preparation of enantiomerically pure p-substituted phenylethyl hydroperoxides by kinetic resolution and their use as enantioselective oxidants in the asymmetric Weitz–Scheffer epoxidation of E-chalcone. Tetrahedron: Asymmetry, 23(8): 594-601.