Anti-oxidative Potential Survival of E. crassipes Under Oxidative Stress

Received: 22/May/2019, Accepted: 09/Jun/2019, Online: 30/Jun/2019 AbstractThe present study evaluates the kinetic constants of enzymatic assays in crude extracts of roots and shoots of E. crassipes growing in wastewater bodies situated in the trans-Ganges/Yamuna regions of Allahabad. The increased kinetic constants in crude extracts of shoots and roots of E. crassipes showing the ability to protect plants from heavy metal induced oxidative stress by activating multi-defense mechanisms and for better growth in polluted environments. This study demonstrates the phytoremediation potential of E. crassipes for the removal of polluted effluents and their ability to survive under oxidative stress.


INTRODUCTION
Water is an important substance on earth for the survival of living organism i.e. human, plants and animals. Speedy population growth and increasing use of water is likely to become an issue in near future due to limited sources of drinking water [1].
Water pollution is jeopardizing our health. Our drinkable water sources are finite. Less than 1 percent of the earth's freshwater is actually accessible. Challenges only increases by 2050, when global demand for freshwater is expected to be one-third greater by now. Municipal and industrial waste discharges contribute their fair share of toxins contaminating stream, river, lake, ocean, aquifer or other bodies of water, degrading the water quality [2].
Although nature possesses abundant capacity to deal with waste water and even pollution via macrophyte which possess a well-developed fibrous root system and large biomass and has been successfully used in wastewater treatment systems to improve water quality by reducing the levels of organic and inorganic nutrients and help in re-establishing and preserving biological, chemical and physical integrity of water. Conserved wastewater can be applied in the restoration of natural ecosystems, industries and for human consumption [3]. The term -phytoremediation‖ is derived from the Greek word υτο (phyto) = plant and Latin word -remedium‖ = restoring balance, or remediation; involves justifying contaminant concentrations in polluted soils, water or air with naturally occurring plants that have ability to accumulate, crude oil, degrade metals, explosives, pesticides, solvents, , and its derivatives [4] and [5]. Eicchornia crassipes growing in wetlands and flooded fields, their roots play significant role in improving the physico-chemical properties of the wastewater [6], [7] and [8].
The study reports that the increased kinetic constants in crude extracts of shoots and roots of E. crassipes showing the ability to protect plants from heavy metal induced oxidative stress by activating multi-defense mechanisms and for better growth in polluted environments. This study demonstrates the phytoremediation potential of E. crassipes for the removal of polluted effluents and their ability to survive under oxidative stress.
Prayagraj (Allahabad) is situated at 25.45°N 81.84°E in the southern part of Uttar Pradesh at an elevation of 98 metres (322 ft) and stands at the confluence of two rivers, the Ganges and Yamuna. The study covers the urban area as well as the adjoining areas of Allahabad district along with industrial areas. In Allahabad, after an initial survey of waste water bodies were selected as sampling sites from the trans-Ganges/Yamuna regions where E. crassipes was present. Waste water bodies without E. crassipes served as control sampling sites present on trans-Ganges region of Allahabad. The present study also examine the changes occurred in fresh aquatic E. crassipes plants surviving under oxidative stress by estimating the enzymatic kinetic constants and the biochemical parameters in roots and shoots of E. crassipes growing in the polluted aquatic environment of Allahabad city region.
The sample collection and experimental analysis was done from three sampling sites of trans-Ganges/Yamuna areas. The experimental work was performed at the Department of Biochemistry and Biochemical Engineering, SHUATS, Prayagraj (Allahabad) and at the Department of Biochemistry of Allahabad University, Prayagraj (Allahabad).

 Sample Collection:
The free floating E. crassipes (Fig. 1) plants were collected from the sampling sites for further biochemical estimation. The samples were labelled, stored in clean and dry polyethylene bags and transported to the laboratory. The plants were washed in running tap water blotted dry with filter paper. Damage to root and leaf apices were avoided. The samples were refrigerated at 4 o C until used. All assays were carried out at room temperature (25-30 °C).The method followed for enzyme extraction was given by [9].Enzyme extraction was carried out at between 4-6 0 C. The extracts were prepared by grinding the plant sample (5.0g fresh wt) in 10 ml 0.1M phosphate buffer pH 7. The extracts were centrifuged at 15,000g for 30min at 4 0 C in refrigerated centrifuge and the supernatant used for enzyme analysis. Experiments were conducted to determine the effect of substrate concentration on the enzymatic activity of roots and shoots of E. Crassipes. K m and V max value for each enzyme was calculated using the Lineweaver and Burk (1/V vs 1/[S]) plot.

Catalase (CAT):
The enzyme assay was done at five concentrations of H 2 O 2 substrate ranging from 0.2mM to 0.8 mM according to the method described by [10].
Guaiacol peroxidase (POD): K m and V max for POD was done according to the method given by [11]. The enzyme activity was assayed at five concentrations of guaiacol ranging from 0.07 mM to 0.3 mM.
Polyphenol peroxidase (PPO): K m and V max for PPO was done according to the method given by [12]. The enzyme activity was assayed at five concentrations of catechol substrate ranging from 1.7 x10 -3 M to 8.3 x10 -³ M. Estimation of Protein [13].
Statistical Analysis: Each treatment was analyzed with a minimum of 3 replicates and the Standard Deviation (SD) was calculated. All the data reported as MEAN ± SD (Minimum of 3 replicates).
 Catalase (V max and K m ) (1.11.1.6):      From the overall results, it was concluded that the pattern of V max shows the increasing order as CAT> GPX> PPO; the order for K m was as PPO> CAT> GPX observed in both the shoots and roots of E. crassipes present in trans-Ganges/Yamuna sites of Prayagraj. The kinetic constants (V max and K m ) of enzymes in roots from trans-Yamuna sites were higher than in shoots of E. crassipes in comparison to the trans-Ganges sampling sites. K m is an approximate measure of the affinity of the substrate for the enzyme. Large K m value represents low affinity of substrate for enzyme and high K m value is indicative of high affinity for enzymes. The results observed indicates that PPO has maximum K m showing low affinity of substrate followed by CAT and GPX in both shoots and roots of E. crassipes from trans-Ganges/Yamuna sites. V max is the maximum velocity at which the enzyme catalysed a reaction and all its active sites is saturated with substrate. V max for CAT was reported to be maximum followed by GPX and PPO in E. crassipes present in trans-Ganges/Yamuna sites of Prayagraj.
Further work with partially/purified enzymes are required to substantively contribute towards understanding the detailed mechanism of induced oxidative stress in the E. crassipes.
However, a few reports on kinetic data of plants growing in aquatic eco-systems are available The present study is in agreement with the study done by [14] who stated that for CAT, the K m and V max treated samples were 0.72 mM, 0.33mM/ min. mg protein, respectively over the control sample.
[15] study reported that the CAT V max and K m of cabbage leaf was 31.12 µMmin -1 and 25 mM, respectively. In the study of [16] on the effect of crude oil on the lentil shoots the kinetic parameters of the CAT, the K m and V max for the control and treated samples were 1.7 mM, 0.64mMmin -1 mg -1 protein, and 0.72 mM, 0.33mM min -1 .mg -1 protein, respectively were investigated. [17] reported in his study that the K m and V max values of PPO for mulberry leaf and mushroom were 35 mM and 3 Uml -1 , and, 20 mM and 5 Uml -1 , respectively. Polyphenol oxidase activity (PPO) was extensively studied in jackfruit for its role in enzymatic browning. PPO and the phenolic compound play a vital role in defensive mechanism against pest and diseases. For the studies in jack fruit waste, PPO was purified and characterized and its kinetic constant for PPO was found to be 15.82 mM (K m ) and 2182 Uml -1 min -1 (V max ) using catechol as substrate [18].  [29]. PPO is located in the thylakoid membrane of chloroplasts and their phenolic substrates are located in the vacuoles in higher plants In stress condition, the free radical species (forms of active oxygen) are increased, which enhances the activities of these detoxifying enzymes [30]. In the systems involving enzyme, substrate, and metal ion, the metal may react with the substrate, the protein-active groups of enzymes, as well as the enzymesubstrate complex. Information is available on enzyme kinetics (V max and K m values) in the presence of heavy metals. The V max and K m values represent the maximum reaction velocity and the affinity of the enzyme for the substrate, respectively.

IV. CONCLUSION AND FUTURE SCOPE
Summarizing the overall finding of the study, it has been concluded that E. crassipes has the ability of improving the physicochemical factors of water along with the accumulating and removal capacity of the toxic metal substances from sampling sites. E. crassipes reduced the properties of toxic metal stress by increasing the antioxidant enzyme activities and shows the effective changes in biochemical parameters and kinetic constants of enzymes in shoots/roots of E. crassipes present in stressed environment for its better survival in contaminated water bodies at both the trans-Ganges/Yamuna sites of Prayagraj. This study determines that E. crassipes retains all the characteristics of an aquatic plant for use in phytoremediation of wastewater bodies by removing heavy metal contents and improving physicochemical characteristics of the