Studies on the Characterization of Esterase from Different Pisum sativum L. (Fabaceae) Varieties

Pisum sativum L. (Fabaceae) is important in diet due to its fiber content, protein, starch, trace elements and phytochemical substances. Dichlorvos (2, 3-dichlorovinyl dimethyl phosphate) is commonly known as organophosphates belong to the classes of insecticides that are used to control households and stored products insects. The aim of this study was to analyze the kinetic characteristics of esterase enzyme from four different varieties of Pisum sativum L. using qualitative and quantitative method and also the docking position of esterase with Dichlorvos by insilico method. According to our study the total activity was highest for Arka Nirmala and Arka Priya varieties. The specific activity was highest for Arka Mayur variety. Km and Vmax, were found to be higher for Arka Uttam. The optimum temperature and pH for all the varieties were found to be same, 40°C and 7.5 respectively. The inhibitor studies showed Arka Nirmala was more sensitive to inhibitor. From the fitness and interaction profile it was found that the inhibitor dichlorovos is an effective pesticide which can be used to inhibit esterase activity. It has one hydrogen bond of H-S and had an amino acid residue at ARG57.


1.Introduction
Medicinal plants are widely used to treat some disorders and to maintain a healthy state in both modern and traditional medicine [1]. It provides three main benefits: health benefits, financial benefits and society wide benefits. Pisum sativum L. (Fabaceae), as known as green pea or garden pea, is important in every day diet due to its fiber, protein, starch, trace elements and many phytochemical contents [2]. Many studies proved that it has antibacterial, antidiabetic, antifungal, anti-inflammatory, anti-hyper cholesterolemia, antioxidant and anticancer properties [2,3].
Plant esterases are involved in food spoilage. The significant functions of these esterases also include in metabolism and subsequent detoxification of many agrochemicals and pharmaceuticals [4,5]. Thus, the esterases catalyze the hydrolysis of aliphatic and aromatic esters and have been widely studied because of their metabolic functions, in flavor development and role in the breakdown of the insecticides [6].
Dichlorovos (2, 3-dichlorovinyldimethylphosphate) referred as organophosphates which belongs to insecticides. Therapeutically, dichlorovos is used as a fumigant and to treat a variety of parasitic worm infections in dogs, livestock and humans [7,8].
Using docking analysis the targeted protein (esterase) and the ligand (Dichlorovos) was conducted to analyze the fitness and the interaction with each other in the form of energy. This interaction could be used as the pharmaceutical approach for drug production [9,10,11].
The aim of this study is to find the different kinetic characteristics of esterase and also the best fit between dichlorovos inhibitor and the esterase.

Preparation of the enzyme extract
Four varieties of Pisum sativum L. were used: Arka Mayur, Arka Nirmal, Arka Uttam and Arka Priya. 5g of Pisum sativum L. (from GKVK, University of Agricultural Sciences, Bengaluru, India) were weighed and homogenized using mortar and pestle by adding 0.05M chilled phosphate buffer 100 mL (pH =7). The homogenate was centrifuged for 10 min at 10000 rpm in a refrigerated centrifuge and was collected in beaker maintained at low temperature. The volume of the supernatant containing crude enzyme was measured.
For qualitative analysis, FC method and UV-VIS spectrophotometry was used: molybdenum in phosphomolybdate tungstate complex is reduced by tyrosine and tryptophan residues of protein to form blue color complex. Its blue color is enhanced by the presence of copper ions. The protein content obtained can be used for the determination of specific activity of esterase enzyme.
For quantitative analysis, different parameters such as total protein concentration, Km and Vmax, optimum pH and temperature, activation energy and specific activity were analyzed.
Total protein content was done using 25 mL of substrate and appropriate buffer and diluted enzyme was added and incubated for specific time period, the reaction was arrested by adding diazo blue lauryl sulphate (DBLS) reagent and absorbance was read at 600nm.
An inhibition study was carried by determining the esterase enzyme activity against different concentrations of dichlorvos (0.2mM to 1 mM) as inhibitor.

Estimation of the total activity of esterase
Total activity of the enzyme is calculated at different time intervals and it is based on the reaction between substrate and enzyme. It is measured in terms of IU.

Estimation of specific activity of esterase
Specific activity gives a measurement of enzyme purity in the mixture. It is calculated by dividing the total activity with total protein and it is measured in terms of IU/mg.

Estimation of Km and Vmax
Km is defined as the substrate concentration at velocity which is half from the final velocity that is Vmax. Different volumes of 0.3 mM substrate solution was taken in different tubes and made up to 5mL with 0.5M phosphate buffer (pH=7). Diluted enzyme (1:20) is taken and 1mL is added to tubes at different time intervals and incubated for 15 min. After this, DBLS is added and reaction is arrested and again incubated for 15 min for color development. Km and Vmax are measured in terms of mM and μm/min respectively.

Estimation of optimum pH
Phosphate buffer, Citrate buffer and Tris buffer was added in different tubes along with 1mL of enzyme and incubated for 1 h. 0.5mL of substrate was added and again incubated for 20 min; DBLS was added to arrest the reaction and again incubated for 20 min.

Estimation of Optimum temperature and activation energy
To the substrate (5.0 mL), 0.5 mL enzyme was added and incubated at different temperatures 20°, 25°, 30°, 40°, 45°C for 20 min. Reaction was arrested using DBLS. Then tubes were kept at room temperature for 15 min. Absorbance was read at 600nm.

Estimation of IC 50 value of the inhibitor
IC is expressed as an inhibitor concentration at which 50 % of the enzyme is inhibited. To all tubes, 2.5 mL of different concentrations of inhibitor (0.2 mM, 0.4mM, 0.6mM, 0.8mM and 1 mM) and 0.5 mL of enzyme were added. The tubes were incubated at 10°C for 10 min. Then 2.5 mL of different concentrations of substrate were added and incubated for 10 min at room temperature. The reaction was stop by adding 1 mL of DBLS. All the tubes were incubated for 15 min at room temperature. The absorbance was read at 600 nm.

In silico analysis
The protein data bank (PDB) was used to obtain the three-dimensional structure of the macromolecule. The downloaded proteins were viewed in Py-Mol viewer (version 2.3.4) [12,13]. Ligand (Dicholorvos), used for the study was constructed using ChemSketch. The constructed ligands were optimized to add the hydrogen bonds and the obtained structures were saved in mol for docking analysis. Docking studies were conducted using iGEMDOCK software [14]. The best binding pose, the binding affinity and the total binding energy values were saved in the output folder. The saved files were visualized in Py-Mol viewer [15,16].

Figure 1. Protein concentration of samples
It was found that all four samples of Pisum sativum L. have different total protein content. Arka Uttam had the highest protein (4.0 mg % extract) content in the extract, followed by Arka Priya (3.2 mg %), Arka Mayur (2.4mg %) and Arka Nirmala (1.6mg %). The concentration of the protein determines the quality of the Pisum sativum L. and it is strongly influenced by plant genetics and growing conditions [17].  Figure 2, it was found that all different varieties had various total activities for the same enzyme. Among four, Arka Nirmala and Arka Priya showed highest total activity of 0.10 IU, whereas Arka Uttam and Arka Mayur has very less total activity of 0.02 IU. More total activity determines the quality of the Pisum sativum L. and it is strongly influenced by many factors [17] Estimation of specific activity of esterase Specific activities of esterase enzyme from four different sources were given in Figure 3. It was seen that all the varieties has almost near specific activities. But comparatively, Arka Mayur showed a slight highest specific activity of 0.008 IU/mg, which was followed by Arka Nirmala (0.006 IU/mg), Arka Uttam (0.005 IU/mg) and Arka Priya (0.003 IU/mg). Specific activity will be more accurate than the total activity. More specific activity determines the amount of esterase present in the solution [18].   Figure 6, the optimum pH for esterase enzyme in all four different samples was found out to be 7.5. The activity of the enzyme depends on pH of the solution. Most of the enzymes are active at neutral pH that is between 6.5 -7.5 [19].  Figure 7, the optimum temperature was found the same for all four samples. The optimum temperature of esterase enzyme for all four different varieties was 40°C [19]. preceded by Arka Mayur having 10 calories. Arka Uttam and Arka Priya had similar activation energies of 7.0 calories and 7.2 calories respectively. Lower the activation energy higher is its rate of reaction [18].   Estimation of fitness and interaction of inhibitor with esterase:   Table 1 and Figure 14, the fitness and the interaction profile of inhibitor dichlorovos with esterase enzyme can be analyzed. It was found that the energy with which the inhibitor binds to esterase was around -68.18 kcal/mol. The Van der Wall's force of attraction between them was found to be as -40.37 kcal/mol, but there was absence of electrostatic force of attraction. One amino acid, ARG 57 which was present at the catalytic center was inhibited by dichlorovos. One hydrogen bond predominantly found (H-S bond) with the bond energy -9.2 kcal/mol.

4.Conclusions
Among the four samples used for analysis, it was found that Arka Uttam variety had the highest concentration of protein approximately of about 4 mg in 100 mL extract.
The total activity was similar in two samples, Arka Nirmala and Arka Priya, about 0.010 IU. But the specific activity was highest for Arka Mayur (0.08 IU/mg). Km and Vmax, the two kinetic characteristics, were found to be higher in Arka Uttam having the values of 0.072mM and 3.3μm/min respectively. The optimum temperature and optimum pH for all the samples were found to be same, 40°C and 7.5 respectively. From the inhibitor studies it was found that, Arka Nirmala was more sensitive to Inhibitor at IC 50 levels. From the fitness and interaction profile it was found that the inhibitor dichlorovos is an effective pesticide which can be used to inhibit esterase activity. It has one hydrogen bond of H-S and ARG57, an amino acid residue is present at the catalytic centre.