Peroxidases are oxidoreductases that catalyze a variety of reactions such as reduction of peroxides and oxidation of a variety of organic and inorganic compounds. They are the second largest class of enzymes applied in biotechnological processes (Twala et al., 2020). It has emerged as an excellent biocatalyst for organic bio-transformation reactions (Singh et al., 2013). Peroxidases from different sources are unique in their characteristics and they are widely applicable in industries and biotechnology. Although, peroxidases are widely distributed in the biosphere, plant sources for the enzyme have not been fully explored. Nevertheless, peroxidases have been reported in plants such as horseradish, papaya (Carica papaya), banana (Musa paradisiacal), bare (Acorus calamus) and ginger (Zingiber officinale) among others. Although many works have been done in search of alternative source of peroxidase other than the commercial source, horseradish roots, nonetheless, turmeric has not been investigated for the presence of peroxidase. The search for an alternative source for this enzyme cannot be over emphasized, due its versatility and numerous applications which include transformation of pollutants to non-toxic metabolites, biodegradation of plastics, bioremediation of waste water containing phenols, cresols, and chlorinated phenols, for bio-pulping and bio-bleaching, decolourization and degradation of synthetic textile dyes, removal of peroxide from materials such as food stuffs and industrial wastes, as biosensors diagnostic kits, in ELISA for labeling an antibody and synthesis of various aromatic chemicals, among others. This work investigated the catalytic properties of purified peroxidase from rhizome of turmeric (Cucuma longa L.) (ClP) for scientific documentation.
Enzyme purification from biological sources, most importantly, plants is an area of research in biochemistry which has attracted much interest recently. This is due to enzyme numerous applications and plant ubiquitousness (Ilesanmi and Adedugbe, 2023). Peroxidase as an industrial enzyme with vast deployment in biotechnology, requires fast purification technique, of such is aqueous two-phase partitioning system (ATPS) employed in this work as the major purification step for turmeric peroxidase. ATPS are biphasic systems for extraction, concentration, purification and separation by partitioning into two immiscible aqueous phases, composed of polymers and salts. It is a well-known technique that has been currently employed by researchers in the area of enzyme technology in both laboratory and industrial enzyme production and recovery. ATPS method of purification is carried out in mild conditions, thereby enzymes and biomolecules to be isolated are not denatured in the process, and also, they are simple to be carried out, speedy, cost effective and accurate. This purification system combines purification and concentration of the enzyme, consequently reducing the lengthiness and cumbersomeness of purification protocol as compared to conventional procedures. ATPS was carried out as a single purification step on the turmeric peroxidase. The specific activity of purified ClP was estimated to be 44,929 ± 0.3514 Units/ mg protein with purification fold of 5.02 and a percentage yield of 51.25%. The yield and purification fold obtained for turmeric peroxidase is as a result of removal of unwanted materials and polyphenols in the plant material during the purification process. The purification parameters can be greatly improved by optimizing the process. Increase in peroxidase activity is usually associated with environmental stress such as drought on plants. Lower purification fold of 2.18 was obtained for peroxidase from Ipomea palmetta. The ease of purification of this enzyme, makes it an economically viable source for production of commercial peroxidase.
Molecular weight determination is important and varies according to sources of the enzyme, tissue expressions patterns and possibly covalent aggregations and/ or modifications during purification procedures. Peroxidase from different sources have similar or / and varied molecular weights (Khatun et al., 2012). The post ATPS obtained in this research was subjected to SDS PAGE to ascertain purity of this enzyme. Non-denaturing polyacrylamide gel electrophoresis (PAGE) analysis carried out in this work gave a single band estimated to be 69 ± 0.2 kDa. The enzyme is monomeric in nature and likely pure, this is revealed by the single band obtained. This molecular weight was further corroborated on mass spectrometry analysis which gave a similar result of 72.1 ± 0.3 kDa as the estimated molecular weight. Mass spectrometry is noted for accurate and precise molecular weight determination, stemming from its superior sensitivity for detecting low abundance molecules. The molecular weight of purified turmeric peroxidase obtained in this work is higher than peroxidase in ginger (Zingiber officinale) 42 kDa, Sunflower roots; 47 kDa, and Ziziphus jujuba fruit 56 kDa. However, it is similar to molecular weight of peroxidase isolated from white cabbage (Brassica oleracea var. capitata f. alba); 73.2 kDa (Erdem et al., 2014). Furthermore, in a study, Moneera et al. (2019) obtained a similar molecular weight of 68 ± 3 kDa on SDS-PAGE for peroxidase from date palm (Phoenix dactylifera). Generally, in literature, molecular weight of plant peroxidases varies widely between 20 to 70 kDa, this variation may be due to differences in amino acid sequence or degree of glycosylation.
Substrate specificity is another important property of enzyme that gives information on the strength and selectiveness of binding to substrate molecule from a range of chemically similar compounds. Furthermore, it is a hallmark of enzyme catalysis whilst, conformational change. Peroxidases are known to exhibit group specificity toward phenolic compounds. The phenolic compounds are classified as mono-phenols, di-phenols and tri-phenolic substrates. Natural plant phenolic compounds such as pyrogallol, guaiacol and catechol are known to be excellent electron donors for plant peroxidases (El-khonezy et al., 2020). Substrate specificity of purified turmeric peroxidase was investigated to provide information on preferred substrate of the enzyme. Plant peroxidases are presumed to have broad substrate specificity spectrum, however, catechol, a di-phenol emerged with the highest activity among the substrates tested. The other substrates include pyrogallol, L-DOPA and O-dianisidine which are a tri-valent phenol, bi-valent phenol and mono-valent phenol respectively. This finding is distinct due to broad substrate specificity exhibited by most documented plant peroxidases. This is advantageous in enzyme identification and separation from millions of similar enzymes.
Studies on effect of temperature and enzyme stability at varying temperature play important role on enzyme catalysis, particularly, structural and functional dynamics of peroxidase ref. The optimum temperature for purified turmeric peroxidase obtained was 60 ºC. Date palm peroxidase and Jatropha curcas has also the optimum temperature of 60 ºC. Moringa oleifera leaves was reported to have the optimum temperature of 50 ºC similarly for ginger rhizomes, however the highest activity was reported for turnip peroxidase at 30 ºC. Studies on oil palm tree leaves peroxidase revealed optimum temperature of 50 ºC and 55–60 ºC for jack fruit peroxidase which was similar to what was obtained for peach fruit. Date palm peroxidase, red beet (Beta vulgaris), spinach (Spinacia oleracea) and Jatropha curcas were reported to have optimum temperature of 60 ºC and more. Similarly, rubber tree peroxidase was stable in the range of temperature 0–60°C and has more than 50% of its activity retained at 70°C even after 30 min of incubation. Highest activity was also demonstrated at 40°C for Pistia stratiotes leaf peroxidase, however, a sharp decline was observed as temperature increases. Nonetheless, ginger (Zingiber officinale) peroxidase activity and carrot (Daucus carota) peroxidase activity were maximal at 50°C. Ginger peroxidase also retained up to 66% of activity for 1 hr at 70°C whereas, it was reported for carrot peroxidase to be thermally stable at 30°C, retaining its maximum activity even at 1 hr of incubation. The activity of the turmeric peroxidase was stable over a broad range of temperatures (10 ºC – 60 ºC). Highest stability of the enzyme was found to be 30°C, although, up to 57% of activity of the enzyme was obtained at 70°C even after 30 mins of incubation. The decline in activity observed at and above 80°C may be as a result unfolding of the enzyme protein globular structure induced by high temperature, leading to loss of peroxidase activity. Variations in optimum temperature of peroxidases may be influenced by differences in purification method and even source of the enzyme. Enzyme stable at high temperatures may find more applications in industries where, their processes are mostly carried out at high temperatures.
Furthermore, enzymes catalyze chemical reactions by lowering activation energy barriers in order to convert substrate molecule to products. Activation energies are characterized by the minimum energy required to cause a reaction process to start by breaking bonds in substrates and are determined from experimental rate constants that are measured at different temperatures. Enzymes act by lowering the activation energy, thereby increasing the rate of reaction. Activation energy of purified turmeric peroxidase was estimated to be 3.67KJ/ mol. This compared well with horse radish peroxidase (HRP) which was calculated to be 3.5 KJ/ mol, similarly, 7.97 KJ/ mol for peach fruit peroxidase. Some other workers have also reported activation energies for peroxidases from different plant sources. Higher activation energy of 206.40 KJ/ mol was reported for jack fruit, 84.79 ± 2.2 KJ/ mol for plums peroxidase, 43.89 KJ/ mol for Ziziphus jujuba fruit peroxidase and 32 KJ/ mol for carrot (Daucus carota). These disparities in activation energies indicate that plant peroxidases work at different rate and ClP is quite similar to HrP in bringing the reaction to completion.
Formation of enzyme substrate complex which is important in the initiation of catalysis is greatly influenced by the conformation of enzyme active site. The ionization states of amino acids which can be affected by pH variation at the active site are of immense importance. A change in pH affects polarity of the amino acids at the active site causing rearrangements in their conformation at the site. The effect of this rearrangement at the active site affects enzyme activity significantly. The optimum pH obtained for purified turmeric peroxidase is pH 8.0. pH stability over this range of pH (6.0- 8.5) was exhibited by the enzyme. The optimum pH obtained in this work agrees with zucchini heads peroxidase (Al-Madhagi et al., 2023), similarly, the optimum pH reported for turnip peroxidase was pH 8.0. Moreover, generally, peroxidases are highly stable in pH range of 5.0 to 9.0. Although, Thitikorn et al. (2013) reported the effect of pH on peroxidase activity obtained from rubber tree (Havea brasiliensis) cell suspension to be pH 5.0, the enzyme activity was still retained between 5.0–10.0, nonetheless pH 6.5 was reported for basil peroxidase and carrot (Daucus carota) peroxidase. Furthermore, pH 5.0 was also reported for oil palm tree peroxidase and pH 5.5 was reported for jack fruit peroxidase. Nevertheless, ginger (Zingiber officinale) peroxidase showed maximum activity pH 6.0- 7.5. The notable wide variations in optimum pH for enzyme activity for plant peroxidases, could be due to different enzyme sources, climatic conditions, soil conditions and environmental stresses among other factors. The enzyme could be promising in applications requiring alkaline pH.
The contribution of metal ions in the maintenance of tertiary and quaternary structures of enzymes cannot be overemphasized. Metals bind through coordinated links which may either maintain, activate or inhibit the activity of enzyme. Peroxidase activity is also dependent on nature of metal ion binding. Turmeric peroxidase was significantly activated by most of the metal ions tested, namely; chloride of Na+, K+, Ca2+ and sulphate of Cu2+ and Zn2+except chloride of Ba2+ and NH4+ which has inhibitory effect. The enhanced effect of these metals mostly monovalent and divalent cations on turmeric activity may be due to stabilization of conformation of the tertiary structure of the enzyme induced by binding. Similar results were exhibited by Ginger peroxidase which was reported to be activated by most divalent cations tested except Hg2+ and Cu2+ (El-khonezy et al., 2020) whereas, Cu2+ activates turmeric peroxidase. The inhibitory effect exhibited by the metals on turmeric peroxidase activity may be as a result of strong binding interaction between amino acids at the enzyme active site and the metals leading to disruption of the enzyme structure, hence, loss of activity.