Enzymatic defence mechanisms in Sapindus mukorossi and Acacia concinna: A Michaelis Menten model approach

Enzymatic defence mechanisms in Sapindus mukorossi and Acacia concinna: A Michaelis Menten model approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enzymatic defence mechanisms in Sapindus mukorossi and Acacia concinna: A Michaelis Menten model approach Rushita Parmar, Vaishali Varsani, Dushyant Dudhagara, Sandip Gamit, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7436731/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 30 You are reading this latest preprint version Abstract Sapindus mukorossi (soapnut) and Acacia concinna , belonging to the families Sapindaceae and Leguminosae respectively, are medicinal plants known for their high saponin content as part of their adaptation and defence strategies against environmental stresses. This study investigates and compares the enzymatic antioxidant defence responses of these two saponin rich species, focusing on key enzymes involved in plant stress role in plant adaptation. Enzymatic assays displayed notable differences in the activities of catalase, peroxidase, and polyphenol oxidase; Acacia concinna possessed higher overall enzymatic activity, while Sapindus mukorossi possessed higher polyphenol oxidase activity reflecting their ecological adaptation and biochemical resilience. The comparative kinetic profiling highlights the enzymatic adaptability of both species under oxidative stress, emphasizing the ecological and biochemical roles of saponins in defence. Linear Discriminant Analysis (LDA), which captured 96.53 percent of the total variation, proved a clear isolation based on enzymatic profiles. Post hoc analysis confirmed statistically significant differences (p ≤ 0.05) in enzyme activity between the two species. These findings provide insights into the metabolic resilience of saponin-rich plants and contribute to understanding plant defence mechanisms in stressful environments. Biological sciences/Biochemistry Biological sciences/Plant sciences Protein Catalase Peroxidase Polyphenol oxidase kinetic defence Adaptation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Sapindus mukorossi , also known as Aritha or Ritha, is a soap nut native to India. Its pericarp is used for washing clothes and cleaning due to its soapy lather. The fruit contains mucilage, sugars, and 11.5% saponins, which are glycosides with foaming properties. It produces seven known saponins, two new dammarane type saponins, and four oleanan Ayurveda-based treatments 1 , 2 Acacia concinna , a medicinal plant in tropical rainforests. The plant's fruits are used for purgative, emetic, expectorant, and hair growth, with chemical evaluations focusing on flavonoids and monoterpenoids 3 , 4 . Phytochemicals, such as saponins, can enhance the activation of antioxidant enzymes and neutralize reactive oxygen species (ROS), making them useful in antioxidant therapies for oxidative stress-related conditions, anti-aging, and protective skincare formulations. These compounds can directly scavenge ROS or enhance the body's natural antioxidant Defence system 5 , 6 , 7 . Plants have a powerful antioxidant defence mechanism consisting of enzymes like catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO). These enzymes scavenge reactive oxygen species (ROS) to protect cells from oxidative damage 8 , 9 . Catalase, a key antioxidant enzyme, converts hydrogen peroxide into water and polyphenol oxidase converts phenolic compounds into quinones, which are polymerized to melanin pigment 10 , 11 , 12 , 13 . Catalase (CAT), present in the cytosol, mitochondria, and peroxisomes, dismutates H 2 O 2 into H 2 O and O 2 , and is present in both prokaryotes and eukaryotes 14 . This system helps reduce oxidative damage caused by ROS 9 . The industrial sector uses catalase for a variety of purposes, from producing porous materials for the textile industry to removing excess H 2 O 2 from milk used in the cold pasteurization process to make cheese 13 , 15 . Every cell contains peroxidases, APX and GPX in particular, which catalyse the conversion of H 2 O 2 to H 2 O. Because it uses ascorbate as an electron donor in the first phase of the ascorbate glutathione cycle, APX is considered the most crucial plant peroxidase in H 2 O 2 detoxification 16 . H 2 O 2 gets broken down enzyme GPX with less specificity to electron donor substrates where co-substrates such as ascorbate and phenolic compounds are used as oxidants 17 . Peroxidases are categorized as animal, plant, fungal, and bacterial peroxidases based on their structure and amino acid arrangement. Variation in amino acid sequences are the cause of these peroxidase species 18 . The peroxidase enzyme is essential for Defence mechanisms, hormone modulation, however, lignin production, and reducing the amount of indole acetic acid in fruits and vegetables 19 , 20 , 21 . Phenolic compound oxidation by polyphenol oxidase (PPO) causes enzymatic browning in food processing and post-harvest physiology of plant products 22 . PPO plays a role in defence mechanisms against insect and plant pathogen attacks, preventing bacterial growth and acting as polyphenolic barriers. It alters plant proteins, reducing nutritional availability to herbivores or intruders 23 . The effective enzymatic antioxidant Defence of plants relies on enzymes like catalase, peroxidase, superoxide dismutase, and polyphenol oxidase 24 . Single-substrate kinetic mechanisms are the first steps in evolutionary processes, while enzymes can catalyse complex reactions using sequential and non-sequential mechanisms. Sequential mechanisms involve a displacement reaction between substrates, while random mechanisms have no obligatory binding sequence, making the reaction more complex 25 , 26 . Research on plant defence and metabolism often focuses on key enzymes such as catalase, peroxidase, and polyphenol oxidase, The comparative kinetic profiling of catalase, peroxidase, and polyphenol oxidase in Sapindus mukorossi and Acacia concinna highlights their enzymatic adaptability under oxidative stress conditions. These responses underscore the critical role of saponins in modulating enzyme activity, enhancing stress tolerance, and contributing to the plants’ ecological fitness. By applying the Michaelis-Menten kinetic model, this study aims to elucidate the catalytic efficiency and substrate specificity of these key enzymes, thereby providing deeper insights into the biochemical and ecological defence strategies of saponin-rich species. These findings not only underscore the importance of CAT, POD, and PPO in stress adaptation but also highlight the ecological significance and pharmaceutical potential of Sapindus mukorossi and Acacia concinna as model plants for studying plant defence responses. 2. Material and Methods 2.1 Collection of Samples The fruits of Sapindus mukorossi and Acacia concinna used in this enzyme study were procured from a local market in Junagadh, Gujarat, India. 2.2 Quantification of total protein Protein estimation was determined using the method described by Folin lowery using bovine serum albumin as the standard 27 . 2.3 Enzyme kinetic modelling 1g of Fruits powder was mixed with in 10 mL of extraction buffer (0.1 M phosphate buffer + 0.5 mM EDTA, pH adjusted to 7.5). The sample was centrifuged at 15000 rpm for 20 min. Supernatant was collected and utilized for the enzyme assays 28 . 2.3.1 Analysis of catalase activity The OD value were evaluated by taking 1500µl of 100mM phosphate buffer with pH 7.0, 500µl of (5 mM to 25 mM) H 2 O 2 , 500 µl of milli-Q and enzyme supernatant in the quartz cuvette the rate of H 2 O 2 breakdown was determined for 0, 5, 10, 15 and 20 minutes at an interval between the samples in the ultraviolet light spectrum of the spectrophotometrically at 240nm. The OD value was also registered in the blank, without the enzyme’s supernatant. The catalase activity was determined spectrophotometrically at room temperature by monitoring the decrease in absorbance resulting from the decomposition of H 2 O 2 at 240 nm. The enzyme activity was expressed in µM H 2 O 2 (ɛ = 39.4 mM − 1 cm − 1 ) oxidized min − 1 mg − 1 protein 29 . 2.3.2 Analysis of Peroxidase activity The OD value were evaluated by taking 1000µl of 100mM phosphate buffer with pH 6.1, 500µl of (92 mM to 100mM) Guaiacol, 400µl of milli-Q 500µl of (6 mM to 14 mM) H 2 O 2 and enzyme supernatant in the quartz cuvette, the rate of Guaiacol and H 2 O 2 decomposition was estimated for 0, 5, 10, 15 and 20 minutes at time interval between the samples in the ultraviolet light spectrum of the spectrophotometrically at 470 nm. The OD value was also registered in the blank, without the enzyme supernatant. The increase in the absorption caused by oxidation of guaiacol by H 2 O 2 (ɛ = 26.6 mM − 1 cm − 1 ) 30 . 2.3.3 Analysis of Polyphenol oxidase activity The OD value were evaluated by taking 2900µl of (100 mM to 500 mM) Catechol in the 10mM phosphate buffer with pH 6.0, 100µl milli-Q and enzyme supernatant in the quartz cuvette the rate of Guaiacol and H 2 O 2 decomposition was estimated for 0, 5, 10, 15 and 20 minutes at time interval between the samples in the ultraviolet light spectrum of the spectrophotometrically at 490 nm. The OD value was also registered in the blank, without the enzyme supernatant. The polyphenol oxidase activity was determined by measuring the increase in absorbance resulting from the oxidation of catechol (ɛ = 1.0 mM − 1 cm − 1 ) at 490 nm spectrophotometrically 31 . Specific activity (U mg − 1 of protein) S.A = \(\:\:\frac{\text{C}\text{h}\text{a}\text{n}\text{g}\text{e}\:\text{i}\text{n}\:\text{O}\text{D}\:\text{p}\text{e}\text{r}\:\text{m}\text{i}\text{n}\text{u}\text{t}\text{e}}{\text{M}\text{o}\text{l}\text{a}\text{r}\:\text{e}\text{x}\text{t}\text{i}\text{n}\text{c}\text{t}\text{i}\text{o}\text{n}\:\text{c}\text{o}-\text{e}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{t}\:\text{o}\text{f}\:\text{e}\text{n}\text{z}\text{y}\text{m}\text{e}\:\left({\upvarepsilon\:}\right)\:\times\:\text{V}\text{o}\text{l}\text{u}\text{m}\text{e}\:\text{o}\text{f}\:\text{e}\text{n}\text{z}\text{y}\text{m}\text{e}\:\text{i}\text{n}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:}\) × Total protein content Where, S.A = Specific activity, Molar extinction co-efficient of enzyme: Catalase = 39.4 U/ \(\:\mu\:\) mols/g, Peroxidase = 26.6 U/ \(\:\mu\:\) mols/g, Polyphenol oxidase = 1.0U/ µ mols/g, 2.4 The Michaelis Menten kinetic equation The kinetic equation in Michaelis-Menten One of the earliest and still useful mathematical models for a straight forward enzyme catalysed reaction 32 , 33 . Michaelis–Menten equation can be expressed as the equation below (Eq. 1 ). \(\:\:\:V=\frac{{V}_{max}\:.\:\left[S\right]\:}{{K}_{m}+\left[S\right]}\) [1] Eq. 1 represents the Michaelis-Menten equation. The relationship between the initial rate of product formation and the concentrations of substrates can be expressed in Eq. 1 . It is essential to calculate the maximal velocity ( Vmax ) and the Michaelis constant ( Km ). The amount of enzyme in the reactor may also have an impact on Vmax . The RK4 method was applied to determine the kinetic parameters. kinetics models of this study on enzyme's affinity for the substrate and its catalytic efficiency. A lower Km indicates a higher substrate affinity, which is necessary for an effective defence response under oxidative stress. Vmax represents the maximum catalytic capacity of the enzyme, reflecting the plant’s potential to rapidly neutralize reactive oxygen species (ROS) during stress conditions. Km is used in the context of plant defence as an indicator of the enzyme's affinity for its substrate. Together, these parameters provide insights into the efficiency, speed, and robustness of the enzymatic antioxidant system, which plays a critical role in enhancing plant resilience and adaptation to biotic and abiotic stressors 34 . 2.5 Statistical analysis: Origin (pro) software, version 2024, MS Excel-2019, Minitab® (version 19.2020.1) was used in the present research study for the conducting the Linear Discriminant analysis as well as Post hoc test constructing. 3. Results 3.1 Total protein contents Figure 1 Total Protein content (mg/g ) in selected plant Species at different concentrations. An analysis was conducted to determine the total protein content in saponin rich fruits ( Sapindus mukorossi and Acacia concinna ) at various concentrations. Figure 1 depicts the varying protein content ranges found in different concentration of saponin rich fruits extracts. Sapindus mukorossi exhibited protein content levels of 0.231 mg/g in 0.02 mg/g, 0.283 mg/g in 0.04 mg/g, 0.420 mg/g in 0.06 mg/g, 0.532 mg/g in 0.08 mg/g and 0.549 mg/g in 0.1 mg/g, of concentration. The protein content of Acacia concinna was determined to be 0.639 mg/g in 0.02 mg/g, 0.885 mg/g in 0.04 mg/g, 0.894 mg/g in 0.06 mg/g, 1.022 mg/g in 0.08 mg/g and 1.259 mg/g in 0.1 mg/g, of concentration. If compare two species Sapindus mukorossi and Acacia concinna maximum protein contents was observed in Acacia concinna 0.1 mg/g (1.259 mg/g) and minimum protein content was observed in Sapindus mukorossi 0.231 mg/g in 0.02 mg/g concentrations. If concentration of fruit extract increase total protein content of both the species also increases shown in (Fig. 1). 3.2 Enzyme assay Three type of enzyme perform in fruits Sapindus mukorossi and Acacia concinna of such as Catalase, Peroxidase, and Polyphenol oxidase. 3.2.1 Catalase activity This scientific investigation examined the catalase activity of Sapindus mukorossi and Acacia concinna fruits extract with different concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg/ml) with respect to different time (0, 5, 10, 15 and 20 minute) interval. In (Fig. 2 a), The catalase activity of Sapindus mukorossi fruits extracts was measured at various concentrations. The activity ranged from 0.357 µmols/g − 1 in 0 minutes to 0.029 µmols/g − 1 in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 0.441 µmols/g − 1 in 0 minutes, 0.094 µmols/g − 1 in 5 minutes, 0.090 µmols/g − 1 in 10 minutes, 0.086 µmols/g − 1 in 15 minutes, and 0.070 µmols/g − 1 in 20 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 0.667 µmols/g − 1 in 0 minutes, 0.180 µmols/g − 1 in 5 minutes, 0.126 µmols/g − 1 in 10 minutes, 0.122 µmols/g − 1 in 15 minutes, and 0.110 µmols/g − 1 in 20 minutes at 0.6 mg/ml fruit extract concentration. (Fig. 2 a), The catalase activity of Acacia concinna (B) fruits extracts was measured at various concentrations. The activity ranged from 0.875 µmols/g − 1 in 0 minutes to 0.024 µmols/g − 1 in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 1.271 µmols/g − 1 in 0 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.347 µmols/g-1 in 0 minutes at 0.6 mg/ml fruit extract concentration. The activity increased to 1.432 µmols/g − 1 in 0 minutes at 0.8 mg/ml fruit extract concentration. The activity reached 1.598 µmols/g − 1 in 0 minutes at 1 mg/ml fruit extract concentration. Compare both fruits species the most significant catalase enzyme activity was recorded in the Acacia concinna fruits extract and minimum observed in Sapindus mukorossi fruits extracts. If compare different time duration the majority the catalase enzyme activity will decreased with increase in incubation time but increase with respect to concentration. 3.2.2 Peroxidase activity In (Fig. 2 b), The peroxidase activity of Sapindus mukorossi (A) fruits extracts was measured at various concentrations. The activity ranged from 0.457 µmols/g in 0 minutes to 0.499 µmols/g − 1 in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 0.623 µmols/g − 1 in 5 minutes, 0.628 µmols/g − 1 in 10 minutes, 0.638 µmols/g − 1 in 15 minutes, and 0.642 µmols/g − 1 in 20 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.516 µmols/g − 1 in 0 minutes, 1.590 µmols/g − 1 in 5 minutes, 1.534 µmols/g − 1 in 10 minutes, 1.538 µmols/g − 1 in 15 minutes, and 1.580 µmols/g-1 in 20 minutes at 0.8 mg/ml fruit extract concentration. The activity was also measured at 1.601 µmols/g − 1 in 0 minutes, 1.638 µmols/g − 1 in 5 minutes, 1.639 µmols/g − 1 in 10 minutes, 1.742 µmols/g − 1 in 15 minutes, and 1.662 µmols/g − 1 in 20 minutes at 1 mg/ml fruit extract concentration. In (Fig. 2 b), The peroxidase activity of Acacia concinna (B) fruits extracts was measured at various concentrations. The activity ranged from 0.977 µmols/g − 1 in 0 min to 1.798 µmols/g − 1 in 20 min at 0.2 mg/ml fruit extract concentration. The activity increased to 1.440 µmols/g − 1 in 0 min, 1.663 µmols/g − 1 in 5 min, 1.692 µmols/g-1 in 10 min, 1.698 µmols/g − 1 in 15 min, and 1.699 µmols/g − 1 in 20 min at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.850 µmols/g − 1 in 0 min, 2.110 µmols/g − 1 in 5 min, 2.210 µmols/g − 1 in 10 min, 2.280 µmols/g − 1 in 15 min, and 2.289 µmols/g − 1 in 20 min at 0.8 mg/ml fruit extract concentration. The activity also increased to 2.451 µmols/g − 1 in 0 min, 2.616 µmols/g − 1 in 5 min, 2.718 µmols/g − 1 in 10 min, 2.840 µmols/g − 1 in 15 min, and 3.762 µmols/g − 1 in 20 min at 1 mg/ml fruit extract concentration. Compare both fruits species the most significant Peroxidase enzyme activity was recorded in the Acacia concinna fruits extract and minimum observed in Sapindus mukorossi fruits extracts. If compare different time duration the majority the Peroxidase enzyme activity will increase with increase in incubation time and various concentration. 3.2.3 Polyphenol Oxidase activity In (Fig. 2 c), The polyphenol oxidase activity of Sapindus mukorossi (A) fruits extracts was measured at various concentrations. The activity ranged from 1.055 µmols/g − 1 in 0 min to 1.382 µmols/g − 1 in 20 min at 0.2 mg/ml fruit extract concentration. The activity increased to 1.302 µmols/g-1 in 0 min, 1.339 µmols/g − 1 in 5 min, 1.350 µmols/g-1 in 10 min, 1.369 µmols/g − 1 in 15 min, and 1.379 µmols/g-1 in 20 min at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.345 µmols/g − 1 in 0 min, 1.349 µmols/g − 1 in 5 min, 1.364 µmols/g − 1 in 10 min, 1.378 µmols/g − 1 in 15 min, and 1.382 µmols/g − 1 in 20 min at 0.6 mg/ml fruit extract concentration. The activity was also observed at 1.758 µmols/g − 1 in 0 min, 1.759 µmols/g − 1 in 5 min, 1.864 µmols/g − 1 in 10 min, 1.968 µmols/g − 1 in 15 min, and 1.979 µmols/g − 1 in 20 min at 1 mg/ml fruit extract concentration. In (Fig. 2 c), The polyphenol oxidase activity of Acacia concinna (B) fruits extracts was measured at various concentrations. The activity ranged from 1.171 µmols/g − 1 in 0 minutes to 1.189 µmols/g − 1 in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 1.224 µmols/g − 1 in 0 minutes, 1.347 µmols/g − 1 in 5 minutes, 1.358 µmols/g − 1 in 10 minutes, 1.337 µmols/g − 1 in 15 minutes, and 1.373 µmols/g − 1 in 20 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.432 µmols/g − 1 in 0 minutes, 1.439 µmols/g − 1 in 5 minutes, 1.442 µmols/g − 1 in 10 minutes, 1.446 µmols/g − 1 in 15 minutes, and 1.452 µmols/g − 1 in 20 minutes at 0.6 mg/ml fruit extract concentration. Compare both fruits species the most significant Polyphenol oxidase enzyme activity was recorded in the Sapindus mukorossi fruits extract and minimum observed in Acacia concinna fruits extracts. If compare different time duration the majority the Polyphenol oxidase enzyme activity will increase with incubation time and various concentration. During stress condition plant produce toxic reactive oxygen species (ROS), this enzyme Catalase, Peroxidase and polyphenol Oxidase neutralized this reactive oxygen species. 3.3 Determination of kinetics Parameter The Michaelis-Menten equation was transformed into linear form using the Lineweaver-Burk plot, a method used for the first time to estimate kinetic parameters, utilizing Eq. 1 to express the plot 35 . $$\:\frac{1}{V}=\frac{{K}_{m}}{{V}_{max\:\:}.\:\:\left[S\right]}+\frac{1}{{V}_{max}}$$ 1 The Michaelis-Menten constant Km , substrate concentration [S], maximum product formation rates, and product formation rates were all examined in the study. The y-axis interception point of a straight line was inverted to calculate Vmax , and the negative reciprocal of the x-intercept was used to calculate Km . The values were utilized in the Microsoft Excel solver for the RK4 method. The number of enzymes added affects Vmax value, but substrate concentration has no effect. Km gauges the substrate-enzyme affinity. Km and Vmax are important parameters in enzyme kinetics because they are strongly impacted by substrate concentration 24 , 36 . Table 1 Determination of enzyme kinetics parameter of selected species. Species name Enzyme Substrate K m (mM) −1 V max (µmol/min − 1 ) Sapindus mukorossi Catalase H 2 O 2 45.4862 10.9769 Peroxidase Guaiacol 100.757 0.12809 H 2 O 2 0.02071 4.56829 Polyphenol Oxidase Catechol 3.45237 1.415428 Acacia concinna Catalase H 2 O 2 32.2093 14.1442 Peroxidase Guaiacol 82.5937 0.0966 H 2 O 2 43.0420 0.48019 Polyphenol Oxidase Catechol 0.31491 4.032258 In this kinetic study, From Table 1 , this study shows a value of V max and K m is varies in various substrates (H 2 O 2, Guaiacol, Catechol) in Catalase, Peroxidase and Polyphenol oxidase enzyme activity of Sapindus mukorossi and Acacia concinna fruit. 3.3.1 Catalase enzyme kinetic model in single substrates (Michaelis-Menten Model) Catalase enzyme activity in H 2 O 2 as substrates with various substrate concentration (5, 10, 15 20 and 25 mM) versus different time (0, 5, 10, 15 and 20 min) in Sapindus mukorossi fruits. In the single substrate model, the Lineweaver burk plot will result in a K m value of 45.4862 mM − 1 and V max value of 10.9769 µmol/min − 1 . Similarly, in Acacia concinna fruit, the K m value will be 32.2093 mM − 1 and the V max value will be 14.1442 µmol/min − 1 . The process's low reaction rate was indicated by a low V max value shown in (Fig. 3). The reaction time for a low-reaction rate process would be longer than that of a high reaction rate process. Regarding the K m value, it indicated the ability of the enzyme to bind to the substrate. A lower K m value would suggest more substrate enzyme binding than in a reaction with a higher K m value. The binding of substrate enzyme was reported to be affecting the reaction rate at negative way. The value of K m obtained in this study is considered quite high compared to other studies from (Table 2). Catalase (CAT) is a crucial antioxidant enzyme that protects plant cells from oxidative damage caused by various stress conditions. 3.3.2 Peroxidase enzyme kinetic model in two Substrate (Random bi–bi model) Peroxidase enzyme activity in Guaiacol and H 2 O 2 as substrates with various substrate concentration (92, 94, 96, 98 and 100 mM and 5, 10, 15, 20 and 25mM) versus different time (0, 5, 10, 15 and 20 min) in Sapindus mukorossi fruits. Applying the Lineweaver burk plot to two substrate models (Guaiacol and H 2 O 2 ) will also result in K m values of 100.757, 0.02071 mM − 1 and V max values of 0.12809, 4.56829 µmol/min − 1 . Similarly, in Acacia concinna fruit, K m values will be 82.5937 and 43.0420 mM − 1 and V max values will be 0.0966, 0.48019 µmol/min − 1 shown in (Fig. 2 a & 2 b). The process's low reaction rate was indicated by a low V max value. The reaction time for a low reaction rate process would be longer than that of a high-reaction-rate process. With respect to the K m value, it indicated the ability of the enzyme to bind to the substrate. A lower K m value would suggest more substrate enzyme binding than in a reaction with a higher K m value. Reaction rate was discovered to be negatively impacted by substrate enzyme binding. The value of K m obtained in this study is considered quite high relative to other studies from Table 2. 3.3.3 Polyphenol oxidase enzyme kinetic model in single substrates (Michaelis-Menten Model) Polyphenol oxidase enzyme activity in Catechol as substrates with various substrate concentration (0.1, 0.2, 0.3, 0.4 and 0.5 mM) versus different time (0, 5, 10, 15 and 20 min) in Sapindus mukorossi fruits. When using the Lineweaver burk plot in a single substrate model, the K m value will be 3.45237 mM − 1 and the V max value will be 1.415428 µmol/min − 1 . Similarly, in Acacia concinna fruit, the K m value will be 0.31491 mM − 1 and the V max value will be 4.032258 µmol/min − 1 shown in (Fig. 5). The process's low reaction rate was indicated by a low V max value. The reaction time for a low reaction rate process would be longer than that of a high reaction rate process. Regarding the K m value, it indicated the ability of the enzyme to bind to the substrate. A lower K m value would suggest that there was more substrate-enzyme binding than in a reaction with a higher K m value. The binding of substrate enzyme was reported to be affecting the reaction rate at negative way. The value of K m obtained in this study is considered quite high compared to other studies from Table 2. In the current investigation, there are several factors that may affect the values of K m and V max in a in specific enzyme activity Two of These elements that were especially significant compared to others are the difference in substrate type. The enzyme activity (U) changed when different substrates were used due to the different amounts of enzyme released during substrate degradation. The value of kinetic constants is heavily influenced by the substrate used in the process. K m and V max provide a functional understanding of how well defence related enzymes utilize their substrates under different stress intensities. Enzymes with low K m and high V max are typically more efficient in rapid and strong defence responses, contributing to a plant’s biochemical resilience and adaptive capacity, current research A high K m implies the enzyme requires a higher substrate concentration to function effectively, which may be beneficial during prolonged or high-intensity stress when substrate accumulation is higher. low V max may reflect a slower but sustained defence response, suitable for long-term protection or in species with moderate metabolic rates 34 . 3.4 Statistical analysis: 3.4.1 Linear Discriminants analysis Linear Discriminants analysis (LDA) is a multivariate statistical technique used to analyze datasets which are arranged into several groups or categories. It expands upon the standard Principal Component Analysis (PCA) by enabling the analysis of several groups at once within a dataset. The Fig. 5 indicated that component 1 explains 96.53 of the total variances in the data, indicating that it captures a significant amount of the variability. Hence, component 1 is the dominant principal component, explaining the majority of the variance in specific enzyme activity Other variables exhibit relatively low loadings on component 2, indicating lesser contributions to the variation. Similarly, Component 1 is characterized by strong positive loadings for Peroxidase and polyphenol oxidase activity that these are closely associated with Component 2. While Catalase enzyme activity demonstrates 3.468 variance negative loading value on Component 2 So, LDA found that the combinations of variables had different strengths of association. (Fig. 6 ) also shows the relationship between a specific enzyme activity of the two species in various time duration 37 . 3.4.2 Post hoc Test Science research frequently employs Post hoc test as a statistical method due to its critical role in addressing alpha-level inflation errors, which increase the likelihood of errors (false positives) resulting from the two group comparisons inherent in research studies 38 . The outcomes derived from post hoc test concerning the examined characteristics revealed significant discrepancies in enzyme activity across plant species at P ≤ 0.05. A statistically notable distinction ( P = 0.05) was noted in Sapindus mukorossi and Acacia concinna , indicating variations in their all three enzymes (catalase, peroxidase, and polyphenol oxidase) displayed significant discrepancies in activity of Sapindus mukorossi catalase (SMC) and Sapindus mukorossi peroxidase (SMP) at P value 0.004399, Acacia concinna catalase (ACC) and Acacia concinna peroxidase (ACP) at P value 0.002157, Sapindus mukorossi catalase (SMC) and Sapindus mukorossi polyphenol oxidase at P value 0.000174 and Acacia concinna catalase (ACC) and Acacia concinna polyphenol oxidase (ACPO). In contrast, when comparing two enzyme of same species the Sapindus mukorossi peroxidase (SMP) and Sapindus mukorossi polyphenol oxidase (SMPO) at p value 0.100903 and Acacia concinna peroxidase (ACP) and Acacia concinna Polyphenol oxidase (ACPO) at p value 0.31313 no statistically remarkable differences were observed. The results from (Fig. 7 ) unequivocally demonstrate that distinct species possess unique enzyme profiles. Among the two distinct species analysed, significant differences were identified between SMC-SMP, ACC-ACP, SMC-SMPO and ACC-ACPO. underscoring significant variations in these defensive responses shown in (Fig. 7 ). 4. Discussion Since antioxidant enzymes eliminate excess free radicals from cells, they are thought to be the most crucial component of cellular Defences. One significant mediator of the cytotoxicity brought on by oxidative stress is thought to be H 2 O 2 . It can diffuse in and out of cells and tissues and is made from almost every source of the oxidative cycle. Aerobic cells are protected from O 2 toxicity and lipid peroxidation by antioxidant Defence enzymes like SOD and CAT. Oxygen and the less reactive H 2 O 2 are produced when SOD combines with the superoxide anion radical. Since CAT and GPx subsequently transform H 2 O 2 into water, SOD may shield cells from the harmful effects of superoxide radicals 39 . This work studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase high specific activity of 25,704 U/mg was obtained. Applying the Lineweaver burk plot using one substrates Michaelis constants ( K m ) and maximum reaction velocities ( V max ) were determined using these substrates at various concentrations. The K m and V max of the purified catalase were found to be 16.2 mM and 2.5 µmol/min, result compare with catalase activity in sapindus mukorossi and acacia concinna fruits Specific activity of catalase 0.667 µmols/g − 1 obtained in sapindus mukorossi fruits and 1.598 µmols/g − 1 in acacia concinna fruits. Apply Michalis mention kinetics models in the catalase activity H 2 O 2 as substrate K m value 45.4862 mM − 1 and V max value of 10.9769 µmol/min − 1 and Acacia concinna fruit, the K m value will be 32.2093 mM − 1 and the V max value will be 14.1442 µmol/min − 1 . The value of K m and V max is obtained in this study is considered quite high compared to black gram seeds 40 . Specific peroxidase activity 2.13 (EU/mg protein) obtained from leaves of rocket ( Eruca vesicaria sbsp. Sativa ). Specific peroxidase activity 1.662 µmols/g − 1 obtained in Sapindus mukorossi and 3.762 µmols/g − 1 Acacia concinna fruit. Compare specific activity of this two fruits maximum obtained in leaves of rocket ( Eruca vesicaria sbsp. Sativa ). The V max and K m values were determined by Lineweaver-Burk plot using Guaicol as a substrates K m 375.74 mM and V max 0.314 µmol/L. compare this result with Peroxidase enzyme activity in Guaiacol and H 2 O 2 as substrates with various substrate concentration. Applying the Lineweaver burk plot to two substrate models (Guaiacol and H 2 O 2 ) will also result in K m values of 100.757, 0.02071 mM − 1 and V max values of 0.12809, 4.56829 µmol/min − 1 . Similarly, in Acacia concinna fruit, K m values will be 82.5937 and 43.0420 mM − 1 and V max values will be 0.0966, 0.48019 µmol/min − 1 if compare result with leaves of rocket ( Eruca vesicaria ) the value of K m is low and V max is high significant result was obtained in selected fruits 41 . Polyphenol oxidase enzyme activity from artichoke ( Cynara scolymus L ) heads, catechol as a substrate. Polyphenol oxidase enzyme specific activity was obtained 10,400 (U/ml min) this activity compare with Sapindus mukorossi fruits extracts was 1.979 µmols/g − 1 and Acacia concinna fruits 1.452 µmols/g − 1 for catechol as a substrate. Applying the Lineweaver burk plot to one substrate models Catechol as a substrate will also result ( K m and V max values were 10.2 mM and 19,662 U/ml min result of artichoke plants leaves compare with sapindus mukorossi fruits K m value will be 3.45237 mM − 1 and the V max value will be 1.415428 µmol/min − 1 . Similarly, in Acacia concinna fruit, the K m value will be 0.31491 mM − 1 and the V max value will be 4.032258 µmol/min − 1 significant K m and V max value obtained in Cynara scolymus L leaves compare to selected fruits 42 . 5. Conclusion The current investigation revealed noteworthy variations among two distinct species of exposed to catalase, peroxidase and polyphenol oxidase enzyme activity. These inspections encompassed the total protein content as well as the specific enzyme activity of catalase, peroxidase, and polyphenol oxidase. Comparative enzymatic profiling revealed that both species exhibit significant catalase and peroxidase activity in Acacia concinna species, polyphenol oxidase activity in Sapindus mukorossi as well as significant protein contents were displayed in Acacia concinna species. Acacia concinna species evolved s uperior enzymatic defence systems that contribute to greater ecological adaptability and tolerance to environmental challenges, compared to Sapindus mukorossi. This underscores the importance of integrating biochemical profiling with enzyme kinetics to understand stress adaptation in selected plant species. Linear discriminant analysis displayed of the total variances in the data, indicating that it captures a significant amount of the variability in enzyme activity. Post hoc test confirmed statistically significant variances in enzyme activity among the species, an important discrepancy significant differences were identified between Sapindus mukorossi and Acacia concinna catalase and peroxidase enzyme and catalase and polyphenol oxidase enzyme underscoring significant variations in these defensive responses in plants. These fruits are ecologically important for sustainable environmental adaptation and ecologically valuable for pharmacological innovation. Declarations 6. Acknowledgments The authors are thankful to the Department of Education, Government of Gujarat, for providing SHODH fellowship. 7. Funding Declaration The authors declare that no funding, any grants, or other forms of financial support were received during this research work. 8. Data availability The datasets generated during and analysed during the current study are available from the corresponding author on reasonable request. 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12:04:51","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":17671,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/7783ac03de33c1fa6606af32.png"},{"id":94019500,"identity":"a2d7c58e-3c16-4943-b39f-17f55aa918cf","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":24312,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/eed83eb9c2ceb740e4204213.png"},{"id":94019501,"identity":"8f4f6a1c-8516-416e-afab-c4ca5c08a9ee","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":18906,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/8653056ef3b5164729a9d3b4.png"},{"id":94019502,"identity":"303239c8-f9ae-411a-a772-7be26acb95ff","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25553,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/64ba4bed20a1a0699a1a5d01.png"},{"id":94019507,"identity":"d21b21e3-c057-4d04-ae80-2394fb7adef4","added_by":"auto","created_at":"2025-10-21 12:04:51","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19380,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/76ada09e35a815c0d367e22e.png"},{"id":94019504,"identity":"53ac676f-f045-49f6-bf48-2815b777418f","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"xml","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131814,"visible":true,"origin":"","legend":"","description":"","filename":"4309fbe0c77441fa8a0de7acc17104ba1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/ede6aa21ce5e09e1459eba77.xml"},{"id":94019506,"identity":"5f21e2e5-843d-41d3-b83e-da6ab028a394","added_by":"auto","created_at":"2025-10-21 12:04:51","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148054,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/ae1b11b17f1a4be9d9547314.html"},{"id":94019468,"identity":"99c2fdde-83f2-49e2-bee9-0aa7713ca3b8","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal Protein content (mg/g\u003c/strong\u003e)\u003cstrong\u003e in selected plant Species at different concentrations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/e318ca9c0a8cb8c9519675d1.png"},{"id":94019469,"identity":"77b4e2ef-de97-4280-9a23-8583600bc03b","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199304,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Catalase enzyme activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSapindus mukorossi \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A) \u0026amp; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcacia concinna \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(B) at different time duration with various concentrations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b) Peroxidase enzyme activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSapindus mukorossi\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (A) \u0026amp; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcacia concinna \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(B) at different time duration with various concentrations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) Polyphenol oxidase enzyme activity in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSapindus mukorossi \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(A) \u0026amp; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcacia concinna \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(B)at different time duration with various concentrations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/063d2e2f87090d3c7143d060.png"},{"id":94019467,"identity":"8cb8d802-397d-4f9a-a1a3-8259d2f1121a","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMichaelis-Menten model for catalase enzyme of species (a) \u0026amp; (b) H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e as a Substrates.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/f136efe2278304f3ab16e12c.png"},{"id":94020231,"identity":"9afb5a92-b732-4961-8e03-6d50463b0c53","added_by":"auto","created_at":"2025-10-21 12:12:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":226577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Random bi–bi model for Peroxidase enzyme of species (a) and (b) Guaiacol\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eas a substrates.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b) Random bi–bi model for Peroxidase enzyme of species (a) and (b) H\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e as a Substrates.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/13b71f5a90e3899033965ded.png"},{"id":94019470,"identity":"c08adbb0-82b7-4c14-8b3f-773fa37d3af2","added_by":"auto","created_at":"2025-10-21 12:04:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":88332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMichaelis-Menten model for Polyphenol oxidase enzyme of species (a) and (b).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/b23a2a6e209c9c3bed3aa087.png"},{"id":94020230,"identity":"896ed783-0891-48f6-8e1c-04311fc29e28","added_by":"auto","created_at":"2025-10-21 12:12:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":109571,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLinear Discriminants analysis of catalase peroxidase and polyphenol oxidase enzyme activity in selected species.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/e9da9bf09633a287360bbfe4.png"},{"id":100615767,"identity":"e398e055-9669-42a9-98e1-5bc7ecade683","added_by":"auto","created_at":"2026-01-19 17:36:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2244538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7436731/v1/bd87c957-5145-4287-9c07-0eca112ae91a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enzymatic defence mechanisms in Sapindus mukorossi and Acacia concinna: A Michaelis Menten model approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cem\u003eSapindus mukorossi\u003c/em\u003e, also known as Aritha or Ritha, is a soap nut native to India. Its pericarp is used for washing clothes and cleaning due to its soapy lather. The fruit contains mucilage, sugars, and 11.5% saponins, which are glycosides with foaming properties. It produces seven known saponins, two new dammarane type saponins, and four oleanan Ayurveda-based treatments \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eAcacia concinna\u003c/em\u003e, a medicinal plant in tropical rainforests. The plant's fruits are used for purgative, emetic, expectorant, and hair growth, with chemical evaluations focusing on flavonoids and monoterpenoids\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePhytochemicals, such as saponins, can enhance the activation of antioxidant enzymes and neutralize reactive oxygen species (ROS), making them useful in antioxidant therapies for oxidative stress-related conditions, anti-aging, and protective skincare formulations. These compounds can directly scavenge ROS or enhance the body's natural antioxidant Defence system \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Plants have a powerful antioxidant defence mechanism consisting of enzymes like catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO). These enzymes scavenge reactive oxygen species (ROS) to protect cells from oxidative damage \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Catalase, a key antioxidant enzyme, converts hydrogen peroxide into water and polyphenol oxidase converts phenolic compounds into quinones, which are polymerized to melanin pigment\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Catalase (CAT), present in the cytosol, mitochondria, and peroxisomes, dismutates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e, and is present in both prokaryotes and eukaryotes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This system helps reduce oxidative damage caused by ROS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe industrial sector uses catalase for a variety of purposes, from producing porous materials for the textile industry to removing excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from milk used in the cold pasteurization process to make cheese\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Every cell contains peroxidases, APX and GPX in particular, which catalyse the conversion of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to H\u003csub\u003e2\u003c/sub\u003eO. Because it uses ascorbate as an electron donor in the first phase of the ascorbate glutathione cycle, APX is considered the most crucial plant peroxidase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detoxification\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e gets broken down enzyme GPX with less specificity to electron donor substrates where co-substrates such as ascorbate and phenolic compounds are used as oxidants\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Peroxidases are categorized as animal, plant, fungal, and bacterial peroxidases based on their structure and amino acid arrangement. Variation in amino acid sequences are the cause of these peroxidase species\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The peroxidase enzyme is essential for Defence mechanisms, hormone modulation, however, lignin production, and reducing the amount of indole acetic acid in fruits and vegetables\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePhenolic compound oxidation by polyphenol oxidase (PPO) causes enzymatic browning in food processing and post-harvest physiology of plant products\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. PPO plays a role in defence mechanisms against insect and plant pathogen attacks, preventing bacterial growth and acting as polyphenolic barriers. It alters plant proteins, reducing nutritional availability to herbivores or intruders\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The effective enzymatic antioxidant Defence of plants relies on enzymes like catalase, peroxidase, superoxide dismutase, and polyphenol oxidase\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Single-substrate kinetic mechanisms are the first steps in evolutionary processes, while enzymes can catalyse complex reactions using sequential and non-sequential mechanisms. Sequential mechanisms involve a displacement reaction between substrates, while random mechanisms have no obligatory binding sequence, making the reaction more complex\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eResearch on plant defence and metabolism often focuses on key enzymes such as catalase, peroxidase, and polyphenol oxidase, The comparative kinetic profiling of catalase, peroxidase, and polyphenol oxidase in \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e highlights their enzymatic adaptability under oxidative stress conditions. These responses underscore the critical role of saponins in modulating enzyme activity, enhancing stress tolerance, and contributing to the plants\u0026rsquo; ecological fitness. By applying the Michaelis-Menten kinetic model, this study aims to elucidate the catalytic efficiency and substrate specificity of these key enzymes, thereby providing deeper insights into the biochemical and ecological defence strategies of saponin-rich species. These findings not only underscore the importance of CAT, POD, and PPO in stress adaptation but also highlight the ecological significance and pharmaceutical potential of \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e as model plants for studying plant defence responses.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Collection of Samples\u003c/h2\u003e\u003cp\u003eThe fruits of \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e used in this enzyme study were procured from a local market in Junagadh, Gujarat, India.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Quantification of total protein\u003c/h2\u003e\u003cp\u003eProtein estimation was determined using the method described by Folin lowery using bovine serum albumin as the standard\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Enzyme kinetic modelling\u003c/h2\u003e\u003cp\u003e1g of Fruits powder was mixed with in 10 mL of extraction buffer (0.1 M phosphate buffer\u0026thinsp;+\u0026thinsp;0.5 mM EDTA, pH adjusted to 7.5). The sample was centrifuged at 15000 rpm for 20 min. Supernatant was collected and utilized for the enzyme assays\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Analysis of catalase activity\u003c/h2\u003e\u003cp\u003eThe OD value were evaluated by taking 1500\u0026micro;l of 100mM phosphate buffer with pH 7.0, 500\u0026micro;l of (5 mM to 25 mM) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 500 \u0026micro;l of milli-Q and enzyme supernatant in the quartz cuvette the rate of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e breakdown was determined for 0, 5, 10, 15 and 20 minutes at an interval between the samples in the ultraviolet light spectrum of the spectrophotometrically at 240nm. The OD value was also registered in the blank, without the enzyme\u0026rsquo;s supernatant. The catalase activity was determined spectrophotometrically at room temperature by monitoring the decrease in absorbance resulting from the decomposition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at 240 nm. The enzyme activity was expressed in \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (ɛ = 39.4 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) oxidized min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e protein\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Analysis of Peroxidase activity\u003c/h2\u003e\u003cp\u003eThe OD value were evaluated by taking 1000\u0026micro;l of 100mM phosphate buffer with pH 6.1, 500\u0026micro;l of (92 mM to 100mM) Guaiacol, 400\u0026micro;l of milli-Q 500\u0026micro;l of (6 mM to 14 mM) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and enzyme supernatant in the quartz cuvette, the rate of Guaiacol and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition was estimated for 0, 5, 10, 15 and 20 minutes at time interval between the samples in the ultraviolet light spectrum of the spectrophotometrically at 470 nm. The OD value was also registered in the blank, without the enzyme supernatant. The increase in the absorption caused by oxidation of guaiacol by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (ɛ = 26.6 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 Analysis of Polyphenol oxidase activity\u003c/h2\u003e\u003cp\u003eThe OD value were evaluated by taking 2900\u0026micro;l of (100 mM to 500 mM) Catechol in the 10mM phosphate buffer with pH 6.0, 100\u0026micro;l milli-Q and enzyme supernatant in the quartz cuvette the rate of Guaiacol and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition was estimated for 0, 5, 10, 15 and 20 minutes at time interval between the samples in the ultraviolet light spectrum of the spectrophotometrically at 490 nm. The OD value was also registered in the blank, without the enzyme supernatant. The polyphenol oxidase activity was determined by measuring the increase in absorbance resulting from the oxidation of catechol (ɛ = 1.0 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 490 nm spectrophotometrically\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpecific activity (U mg\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eof protein)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eS.A\u003c/b\u003e =\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{\\text{C}\\text{h}\\text{a}\\text{n}\\text{g}\\text{e}\\:\\text{i}\\text{n}\\:\\text{O}\\text{D}\\:\\text{p}\\text{e}\\text{r}\\:\\text{m}\\text{i}\\text{n}\\text{u}\\text{t}\\text{e}}{\\text{M}\\text{o}\\text{l}\\text{a}\\text{r}\\:\\text{e}\\text{x}\\text{t}\\text{i}\\text{n}\\text{c}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{c}\\text{o}-\\text{e}\\text{f}\\text{f}\\text{i}\\text{c}\\text{i}\\text{e}\\text{n}\\text{t}\\:\\text{o}\\text{f}\\:\\text{e}\\text{n}\\text{z}\\text{y}\\text{m}\\text{e}\\:\\left({\\upvarepsilon\\:}\\right)\\:\\times\\:\\text{V}\\text{o}\\text{l}\\text{u}\\text{m}\\text{e}\\:\\text{o}\\text{f}\\:\\text{e}\\text{n}\\text{z}\\text{y}\\text{m}\\text{e}\\:\\text{i}\\text{n}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}\\:}\\)\u003c/span\u003e\u003c/span\u003e\u0026times; Total protein content\u003c/p\u003e\u003cp\u003eWhere,\u003c/p\u003e\u003cp\u003eS.A\u0026thinsp;=\u0026thinsp;Specific activity,\u003c/p\u003e\u003cp\u003eMolar extinction co-efficient of enzyme:\u003c/p\u003e\u003cp\u003eCatalase\u0026thinsp;=\u0026thinsp;39.4 U/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003emols/g,\u003c/p\u003e\u003cp\u003ePeroxidase\u0026thinsp;=\u0026thinsp;26.6 U/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003emols/g,\u003c/p\u003e\u003cp\u003ePolyphenol oxidase\u0026thinsp;=\u0026thinsp;1.0U/\u003cem\u003e\u0026micro;\u003c/em\u003emols/g,\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.4 The Michaelis Menten kinetic equation\u003c/h2\u003e\u003cp\u003eThe kinetic equation in Michaelis-Menten One of the earliest and still useful mathematical models for a straight forward enzyme catalysed reaction\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Michaelis\u0026ndash;Menten equation can be expressed as the equation below (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\:V=\\frac{{V}_{max}\\:.\\:\\left[S\\right]\\:}{{K}_{m}+\\left[S\\right]}\\)\u003c/span\u003e\u003c/span\u003e[1]\u003c/p\u003e\u003cp\u003eEq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e represents the Michaelis-Menten equation. The relationship between the initial rate of product formation and the concentrations of substrates can be expressed in Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It is essential to calculate the maximal velocity (\u003cem\u003eVmax\u003c/em\u003e) and the Michaelis constant (\u003cem\u003eKm\u003c/em\u003e). The amount of enzyme in the reactor may also have an impact on \u003cem\u003eVmax\u003c/em\u003e. The RK4 method was applied to determine the kinetic parameters. kinetics models of this study on enzyme's affinity for the substrate and its catalytic efficiency. A lower \u003cem\u003eKm\u003c/em\u003e indicates a higher substrate affinity, which is necessary for an effective defence response under oxidative stress. \u003cem\u003eVmax\u003c/em\u003e represents the maximum catalytic capacity of the enzyme, reflecting the plant\u0026rsquo;s potential to rapidly neutralize reactive oxygen species (ROS) during stress conditions. \u003cem\u003eKm\u003c/em\u003e is used in the context of plant defence as an indicator of the enzyme's affinity for its substrate. Together, these parameters provide insights into the efficiency, speed, and robustness of the enzymatic antioxidant system, which plays a critical role in enhancing plant resilience and adaptation to biotic and abiotic stressors\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical analysis:\u003c/h2\u003e\u003cp\u003eOrigin (pro) software, version 2024, MS Excel-2019, Minitab\u0026reg; (version 19.2020.1) was used in the present research study for the conducting the Linear Discriminant analysis as well as Post hoc test constructing.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Total protein contents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure\u0026nbsp;1 Total Protein content (mg/g\u003c/strong\u003e) \u003cstrong\u003ein selected plant Species at different concentrations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn analysis was conducted to determine the total protein content in saponin rich fruits (\u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e) at various concentrations. Figure 1 depicts the varying protein content ranges found in different concentration of saponin rich fruits extracts. \u003cem\u003eSapindus mukorossi\u003c/em\u003e exhibited protein content levels of 0.231 mg/g in 0.02 mg/g, 0.283 mg/g in 0.04 mg/g, 0.420 mg/g in 0.06 mg/g, 0.532 mg/g in 0.08 mg/g and 0.549 mg/g in 0.1 mg/g, of concentration. The protein content of \u003cem\u003eAcacia concinna\u003c/em\u003e was determined to be 0.639 mg/g in 0.02 mg/g, 0.885 mg/g in 0.04 mg/g, 0.894 mg/g in 0.06 mg/g, 1.022 mg/g in 0.08 mg/g and 1.259 mg/g in 0.1 mg/g, of concentration. If compare two species \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e maximum protein contents was observed in \u003cem\u003eAcacia concinna\u003c/em\u003e 0.1 mg/g (1.259 mg/g) and minimum protein content was observed in \u003cem\u003eSapindus mukorossi\u003c/em\u003e 0.231 mg/g in 0.02 mg/g concentrations. If concentration of fruit extract increase total protein content of both the species also increases shown in (Fig. 1).\u003c/p\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Enzyme assay\u003c/h2\u003e\n \u003cp\u003eThree type of enzyme perform in fruits \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e of such as Catalase, Peroxidase, and Polyphenol oxidase.\u003c/p\u003e\n \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1 Catalase activity\u003c/h2\u003e\n \u003cp\u003eThis scientific investigation examined the catalase activity of \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e fruits extract with different concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg/ml) with respect to different time (0, 5, 10, 15 and 20 minute) interval. In (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), The catalase activity of \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits extracts was measured at various concentrations. The activity ranged from 0.357 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes to 0.029 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 0.441 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes, 0.094 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 0.090 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 0.086 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 0.070 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 0.667 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes, 0.180 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 0.126 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 0.122 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 0.110 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.6 mg/ml fruit extract concentration.\u003c/p\u003e\n \u003cp\u003e(Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea), The catalase activity of \u003cem\u003eAcacia concinna\u003c/em\u003e (B) fruits extracts was measured at various concentrations. The activity ranged from 0.875 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes to 0.024 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 1.271 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.347 \u0026micro;mols/g-1 in 0 minutes at 0.6 mg/ml fruit extract concentration. The activity increased to 1.432 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes at 0.8 mg/ml fruit extract concentration. The activity reached 1.598 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes at 1 mg/ml fruit extract concentration.\u003c/p\u003e\n \u003cp\u003eCompare both fruits species the most significant catalase enzyme activity was recorded in the \u003cem\u003eAcacia concinna\u003c/em\u003e fruits extract and minimum observed in \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits extracts. If compare different time duration the majority the catalase enzyme activity will decreased with increase in incubation time but increase with respect to concentration.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2 Peroxidase activity\u003c/h2\u003e\n \u003cp\u003eIn (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb), The peroxidase activity of \u003cem\u003eSapindus mukorossi\u003c/em\u003e (A) fruits extracts was measured at various concentrations. The activity ranged from 0.457 \u0026micro;mols/g in 0 minutes to 0.499 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 0.623 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 0.628 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 0.638 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 0.642 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.516 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes, 1.590 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 1.534 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 1.538 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 1.580 \u0026micro;mols/g-1 in 20 minutes at 0.8 mg/ml fruit extract concentration. The activity was also measured at 1.601 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes, 1.638 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 1.639 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 1.742 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 1.662 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 1 mg/ml fruit extract concentration.\u003c/p\u003e\n \u003cp\u003eIn (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb), The peroxidase activity of \u003cem\u003eAcacia concinna\u003c/em\u003e (B) fruits extracts was measured at various concentrations. The activity ranged from 0.977 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min to 1.798 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 0.2 mg/ml fruit extract concentration. The activity increased to 1.440 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min, 1.663 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 min, 1.692 \u0026micro;mols/g-1 in 10 min, 1.698 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 min, and 1.699 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.850 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min, 2.110 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 min, 2.210 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 min, 2.280 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 min, and 2.289 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 0.8 mg/ml fruit extract concentration. The activity also increased to 2.451 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min, 2.616 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 min, 2.718 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 min, 2.840 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 min, and 3.762 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 1 mg/ml fruit extract concentration. Compare both fruits species the most significant Peroxidase enzyme activity was recorded in the \u003cem\u003eAcacia concinna\u003c/em\u003e fruits extract and minimum observed in \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits extracts. If compare different time duration the majority the Peroxidase enzyme activity will increase with increase in incubation time and various concentration.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3 Polyphenol Oxidase activity\u003c/h2\u003e\n \u003cp\u003eIn (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), The polyphenol oxidase activity of \u003cem\u003eSapindus mukorossi\u003c/em\u003e (A) fruits extracts was measured at various concentrations. The activity ranged from 1.055 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min to 1.382 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 0.2 mg/ml fruit extract concentration. The activity increased to 1.302 \u0026micro;mols/g-1 in 0 min, 1.339 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 min, 1.350 \u0026micro;mols/g-1 in 10 min, 1.369 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 min, and 1.379 \u0026micro;mols/g-1 in 20 min at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.345 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min, 1.349 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 min, 1.364 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 min, 1.378 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 min, and 1.382 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 0.6 mg/ml fruit extract concentration. The activity was also observed at 1.758 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 min, 1.759 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 min, 1.864 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 min, 1.968 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 min, and 1.979 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 min at 1 mg/ml fruit extract concentration.\u003c/p\u003e\n \u003cp\u003eIn (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), The polyphenol oxidase activity of \u003cem\u003eAcacia concinna\u003c/em\u003e (B) fruits extracts was measured at various concentrations. The activity ranged from 1.171 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes to 1.189 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.2 mg/ml fruit extract concentration. The activity increased to 1.224 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes, 1.347 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 1.358 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 1.337 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 1.373 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.4 mg/ml fruit extract concentration. The activity also increased to 1.432 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 0 minutes, 1.439 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 5 minutes, 1.442 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 10 minutes, 1.446 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 15 minutes, and 1.452 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 20 minutes at 0.6 mg/ml fruit extract concentration. Compare both fruits species the most significant Polyphenol oxidase enzyme activity was recorded in the \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits extract and minimum observed in \u003cem\u003eAcacia concinna\u003c/em\u003e fruits extracts. If compare different time duration the majority the Polyphenol oxidase enzyme activity will increase with incubation time and various concentration. During stress condition plant produce toxic reactive oxygen species (ROS), this enzyme Catalase, Peroxidase and polyphenol Oxidase neutralized this reactive oxygen species.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Determination of kinetics Parameter\u003c/h2\u003e\n \u003cp\u003eThe Michaelis-Menten equation was transformed into linear form using the Lineweaver-Burk plot, a method used for the first time to estimate kinetic parameters, utilizing Eq. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e to express the plot\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\:\\frac{1}{V}=\\frac{{K}_{m}}{{V}_{max\\:\\:}.\\:\\:\\left[S\\right]}+\\frac{1}{{V}_{max}}$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eThe Michaelis-Menten constant \u003cem\u003eKm\u003c/em\u003e, substrate concentration [S], maximum product formation rates, and product formation rates were all examined in the study. The y-axis interception point of a straight line was inverted to calculate \u003cem\u003eVmax\u003c/em\u003e, and the negative reciprocal of the x-intercept was used to calculate \u003cem\u003eKm\u003c/em\u003e. The values were utilized in the Microsoft Excel solver for the RK4 method. The number of enzymes added affects \u003cem\u003eVmax\u003c/em\u003e value, but substrate concentration has no effect. \u003cem\u003eKm\u003c/em\u003e gauges the substrate-enzyme affinity. \u003cem\u003eKm\u003c/em\u003e and \u003cem\u003eVmax\u003c/em\u003e are important parameters in enzyme kinetics because they are strongly impacted by substrate concentration \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDetermination of enzyme kinetics parameter of selected species.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies name\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnzyme\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSubstrate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e(mM)\u003csup\u003e\u0026minus;1\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e (\u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eSapindus\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003emukorossi\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e45.4862\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.9769\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePeroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuaiacol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.757\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.12809\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02071\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.56829\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolyphenol\u003c/p\u003e\n \u003cp\u003eOxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatechol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.45237\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.415428\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcacia\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003econcinna\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatalase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e32.2093\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.1442\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePeroxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGuaiacol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.5937\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0966\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43.0420\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.48019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePolyphenol\u003c/p\u003e\n \u003cp\u003eOxidase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCatechol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.31491\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.032258\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIn this kinetic study, From Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, this study shows a value of \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e is varies in various substrates (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e Guaiacol, Catechol) in Catalase, Peroxidase and Polyphenol oxidase enzyme activity of \u003cem\u003eSapindus mukorossi and Acacia concinna\u003c/em\u003e fruit.\u003c/p\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.1 \u003cstrong\u003eCatalase enzyme kinetic model in single substrates (Michaelis-Menten Model)\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eCatalase enzyme activity in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrates with various substrate concentration (5, 10, 15 20 and 25 mM) versus different time (0, 5, 10, 15 and 20 min) in \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits. In the single substrate model, the Lineweaver burk plot will result in a \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value of 45.4862 mM\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value of 10.9769 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Similarly, in \u003cem\u003eAcacia concinna\u003c/em\u003e fruit, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value will be 32.2093 mM\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value will be 14.1442 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The process\u0026apos;s low reaction rate was indicated by a low \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value shown in (Fig. 3). The reaction time for a low-reaction rate process would be longer than that of a high reaction rate process. Regarding the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value, it indicated the ability of the enzyme to bind to the substrate. A lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value would suggest more substrate enzyme binding than in a reaction with a higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value. The binding of substrate enzyme was reported to be affecting the reaction rate at negative way. The value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e obtained in this study is considered quite high compared to other studies from (Table 2). Catalase (CAT) is a crucial antioxidant enzyme that protects plant cells from oxidative damage caused by various stress conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.2 Peroxidase enzyme kinetic model in two Substrate (Random bi\u0026ndash;bi model)\u003c/h2\u003e\n \u003cp\u003ePeroxidase enzyme activity in Guaiacol and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrates with various substrate concentration (92, 94, 96, 98 and 100 mM and 5, 10, 15, 20 and 25mM) versus different time (0, 5, 10, 15 and 20 min) in \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits. Applying the Lineweaver burk plot to two substrate models (Guaiacol and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) will also result in \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values of 100.757, 0.02071 mM\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e values of 0.12809, 4.56829 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Similarly, in \u003cem\u003eAcacia concinna\u003c/em\u003e fruit, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values will be 82.5937 and 43.0420 mM\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e values will be 0.0966, 0.48019 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shown in (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026amp; \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). The process\u0026apos;s low reaction rate was indicated by a low \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value. The reaction time for a low reaction rate process would be longer than that of a high-reaction-rate process. With respect to the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value, it indicated the ability of the enzyme to bind to the substrate. A lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value would suggest more substrate enzyme binding than in a reaction with a higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value. Reaction rate was discovered to be negatively impacted by substrate enzyme binding. The value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e obtained in this study is considered quite high relative to other studies from Table 2.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.3.3 \u003cstrong\u003ePolyphenol oxidase enzyme kinetic model in single substrates (Michaelis-Menten Model)\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003ePolyphenol oxidase enzyme activity in Catechol as substrates with various substrate concentration (0.1, 0.2, 0.3, 0.4 and 0.5 mM) versus different time (0, 5, 10, 15 and 20 min) in \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits. When using the Lineweaver burk plot in a single substrate model, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value will be 3.45237 mM\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value will be 1.415428 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Similarly, in \u003cem\u003eAcacia concinna\u003c/em\u003e fruit, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value will be 0.31491 mM\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value will be 4.032258 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shown in (Fig. 5). The process\u0026apos;s low reaction rate was indicated by a low \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value. The reaction time for a low reaction rate process would be longer than that of a high reaction rate process. Regarding the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value, it indicated the ability of the enzyme to bind to the substrate. A lower \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value would suggest that there was more substrate-enzyme binding than in a reaction with a higher \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value. The binding of substrate enzyme was reported to be affecting the reaction rate at negative way. The value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e obtained in this study is considered quite high compared to other studies from Table 2.\u003c/p\u003e\n \u003cp\u003eIn the current investigation, there are several factors that may affect the values of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e in a in specific enzyme activity Two of These elements that were especially significant compared to others are the difference in substrate type. The enzyme activity (U) changed when different substrates were used due to the different amounts of enzyme released during substrate degradation. The value of kinetic constants is heavily influenced by the substrate used in the process. \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e provide a functional understanding of how well defence related enzymes utilize their substrates under different stress intensities. Enzymes with low \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and high \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e are typically more efficient in rapid and strong defence responses, contributing to a plant\u0026rsquo;s biochemical resilience and adaptive capacity, current research A high \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e implies the enzyme requires a higher substrate concentration to function effectively, which may be beneficial during prolonged or high-intensity stress when substrate accumulation is higher. low \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e may reflect a slower but sustained defence response, suitable for long-term protection or in species with moderate metabolic rates\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Statistical analysis:\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.1 Linear Discriminants analysis\u003c/h2\u003e\n \u003cp\u003eLinear Discriminants analysis (LDA) is a multivariate statistical technique used to analyze datasets which are arranged into several groups or categories. It expands upon the standard Principal Component Analysis (PCA) by enabling the analysis of several groups at once within a dataset. The Fig. 5 indicated that component 1 explains 96.53 of the total variances in the data, indicating that it captures a significant amount of the variability. Hence, component 1 is the dominant principal component, explaining the majority of the variance in specific enzyme activity Other variables exhibit relatively low loadings on component 2, indicating lesser contributions to the variation. Similarly, Component 1 is characterized by strong positive loadings for Peroxidase and polyphenol oxidase activity that these are closely associated with Component 2. While Catalase enzyme activity demonstrates 3.468 variance negative loading value on Component 2 So, LDA found that the combinations of variables had different strengths of association. (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e) also shows the relationship between a specific enzyme activity of the two species in various time duration \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003ch2\u003e3.4.2 Post hoc Test\u003c/h2\u003e\n \u003cp\u003eScience research frequently employs Post hoc test as a statistical method due to its critical role in addressing alpha-level inflation errors, which increase the likelihood of errors (false positives) resulting from the two group comparisons inherent in research studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The outcomes derived from post hoc test concerning the examined characteristics revealed significant discrepancies in enzyme activity across plant species at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\n \u003cp\u003eA statistically notable distinction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05) was noted in \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e, indicating variations in their all three enzymes (catalase, peroxidase, and polyphenol oxidase) displayed significant discrepancies in activity of \u003cem\u003eSapindus mukorossi\u003c/em\u003e catalase (SMC) and \u003cem\u003eSapindus mukorossi\u003c/em\u003e peroxidase (SMP) at \u003cem\u003eP\u003c/em\u003e value 0.004399, \u003cem\u003eAcacia concinna\u003c/em\u003e catalase (ACC) and \u003cem\u003eAcacia concinna\u003c/em\u003e peroxidase (ACP) at \u003cem\u003eP\u003c/em\u003e value 0.002157, \u003cem\u003eSapindus mukorossi\u003c/em\u003e catalase (SMC) and \u003cem\u003eSapindus mukorossi\u003c/em\u003e polyphenol oxidase at \u003cem\u003eP\u003c/em\u003e value 0.000174 and \u003cem\u003eAcacia concinna\u003c/em\u003e catalase (ACC) and \u003cem\u003eAcacia concinna\u003c/em\u003e polyphenol oxidase (ACPO). In contrast, when comparing two enzyme of same species the \u003cem\u003eSapindus mukorossi\u003c/em\u003e peroxidase (SMP) and \u003cem\u003eSapindus mukorossi\u003c/em\u003e polyphenol oxidase (SMPO) at \u003cem\u003ep\u003c/em\u003e value 0.100903 and \u003cem\u003eAcacia concinna\u003c/em\u003e peroxidase (ACP) and \u003cem\u003eAcacia concinna\u003c/em\u003e Polyphenol oxidase (ACPO) at \u003cem\u003ep\u003c/em\u003e value 0.31313 no statistically remarkable differences were observed. The results from (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e) unequivocally demonstrate that distinct species possess unique enzyme profiles. Among the two distinct species analysed, significant differences were identified between SMC-SMP, ACC-ACP, SMC-SMPO and ACC-ACPO. underscoring significant variations in these defensive responses shown in (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eSince antioxidant enzymes eliminate excess free radicals from cells, they are thought to be the most crucial component of cellular Defences. One significant mediator of the cytotoxicity brought on by oxidative stress is thought to be H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. It can diffuse in and out of cells and tissues and is made from almost every source of the oxidative cycle. Aerobic cells are protected from O\u003csub\u003e2\u003c/sub\u003e toxicity and lipid peroxidation by antioxidant Defence enzymes like SOD and CAT. Oxygen and the less reactive H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are produced when SOD combines with the superoxide anion radical. Since CAT and GPx subsequently transform H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into water, SOD may shield cells from the harmful effects of superoxide radicals\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis work studied catalase, a principal antioxidant enzyme from black gram seeds. Day four sprouted black gram seeds were found to have a significant catalase high specific activity of 25,704 U/mg was obtained. Applying the Lineweaver burk plot using one substrates Michaelis constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) and maximum reaction velocities (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e) were determined using these substrates at various concentrations. The \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e of the purified catalase were found to be 16.2 mM and 2.5 \u0026micro;mol/min, result compare with catalase activity in \u003cem\u003esapindus mukorossi\u003c/em\u003e and \u003cem\u003eacacia concinna\u003c/em\u003e fruits Specific activity of catalase 0.667 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e obtained in \u003cem\u003esapindus mukorossi\u003c/em\u003e fruits and 1.598 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in \u003cem\u003eacacia concinna\u003c/em\u003e fruits. Apply Michalis mention kinetics models in the catalase activity H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrate \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value 45.4862 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value of 10.9769 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e fruit, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value will be 32.2093 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value will be 14.1442 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e is obtained in this study is considered quite high compared to black gram seeds\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Specific peroxidase activity 2.13 (EU/mg protein) obtained from leaves of rocket (\u003cem\u003eEruca vesicaria\u003c/em\u003e sbsp. \u003cem\u003eSativa\u003c/em\u003e). Specific peroxidase activity 1.662 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e obtained in \u003cem\u003eSapindus mukorossi\u003c/em\u003e and 3.762 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003eAcacia concinna\u003c/em\u003e fruit. Compare specific activity of this two fruits maximum obtained in leaves of rocket (\u003cem\u003eEruca vesicaria\u003c/em\u003e sbsp. \u003cem\u003eSativa\u003c/em\u003e). The \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values were determined by Lineweaver-Burk plot using Guaicol as a substrates \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e 375.74 mM and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e 0.314 \u0026micro;mol/L. compare this result with Peroxidase enzyme activity in Guaiacol and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrates with various substrate concentration. Applying the Lineweaver burk plot to two substrate models (Guaiacol and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) will also result in \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values of 100.757, 0.02071 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e values of 0.12809, 4.56829 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Similarly, in \u003cem\u003eAcacia concinna\u003c/em\u003e fruit, \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e values will be 82.5937 and 43.0420 mM\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e values will be 0.0966, 0.48019 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e if compare result with leaves of rocket (\u003cem\u003eEruca vesicaria\u003c/em\u003e) the value of \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e is low and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e is high significant result was obtained in selected fruits\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Polyphenol oxidase enzyme activity from artichoke (\u003cem\u003eCynara scolymus L\u003c/em\u003e) heads, catechol as a substrate. Polyphenol oxidase enzyme specific activity was obtained 10,400 (U/ml min) this activity compare with \u003cem\u003eSapindus mukorossi\u003c/em\u003e fruits extracts was 1.979 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e fruits 1.452 \u0026micro;mols/g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for catechol as a substrate. Applying the Lineweaver burk plot to one substrate models Catechol as a substrate will also result (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e values were 10.2 mM and 19,662 U/ml min result of artichoke plants leaves compare with \u003cem\u003esapindus mukorossi\u003c/em\u003e fruits \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value will be 3.45237 mM\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value will be 1.415428 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Similarly, in \u003cem\u003eAcacia concinna\u003c/em\u003e fruit, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value will be 0.31491 mM\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value will be 4.032258 \u0026micro;mol/min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e significant \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e value obtained in \u003cem\u003eCynara scolymus L\u003c/em\u003e leaves compare to selected fruits\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe current investigation revealed noteworthy variations among two distinct species of exposed to catalase, peroxidase and polyphenol oxidase enzyme activity. These inspections encompassed the total protein content as well as the specific enzyme activity of catalase, peroxidase, and polyphenol oxidase. Comparative enzymatic profiling revealed that both species exhibit significant catalase and peroxidase activity in \u003cem\u003eAcacia concinna\u003c/em\u003e species, polyphenol oxidase activity in \u003cem\u003eSapindus mukorossi\u003c/em\u003e as well as significant protein contents were displayed in \u003cem\u003eAcacia concinna\u003c/em\u003e species. \u003cem\u003eAcacia concinna\u003c/em\u003e species evolved \u003cb\u003es\u003c/b\u003euperior enzymatic defence systems that contribute to greater ecological adaptability and tolerance to environmental challenges, compared to \u003cem\u003eSapindus mukorossi.\u003c/em\u003e This underscores the importance of integrating biochemical profiling with enzyme kinetics to understand stress adaptation in selected plant species. Linear discriminant analysis displayed of the total variances in the data, indicating that it captures a significant amount of the variability in enzyme activity. Post hoc test confirmed statistically significant variances in enzyme activity among the species, an important discrepancy significant differences were identified between \u003cem\u003eSapindus mukorossi\u003c/em\u003e and \u003cem\u003eAcacia concinna\u003c/em\u003e catalase and peroxidase enzyme and catalase and polyphenol oxidase enzyme underscoring significant variations in these defensive responses in plants. These fruits are ecologically important for sustainable environmental adaptation and ecologically valuable for pharmacological innovation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e6. Acknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the Department of Education, Government of Gujarat, for providing SHODH fellowship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funding, any grants, or other forms of financial support were received during this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Data availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePandey, S., Oza, G., Mewada, A., \u0026amp; Sharon, M. Green synthesis of highly stable gold nanoparticles using Momordica charantia as nano fabricator. \u003cem\u003eArch. Appl. Sci. 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Recent developments in discriminant analysis on high dimensional spectral data. \u003cem\u003eChemometrics and Intelligent Laboratory Systems\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(2), 157-173 (1996).\u003c/li\u003e\n \u003cli\u003eJuarros-Basterretxea, J., Aonso-Diego, G., Postigo, \u0026Aacute;., Montes-\u0026Aacute;lvarez, P., Men\u0026eacute;ndez-Aller, \u0026Aacute;., \u0026amp; Garc\u0026iacute;a-Cueto, E. Post-hoc tests in one-way ANOVA: The case for normal distribution. \u003cem\u003eMethodology\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(2), 84-99 (2024).\u003c/li\u003e\n \u003cli\u003eDe Oliveira, F. K., Santos, L. O., \u0026amp; Buffon, J. G. Mechanism of action, sources, and application of peroxidases. \u003cem\u003eFood Research International\u003c/em\u003e, \u003cem\u003e143\u003c/em\u003e, 110266 (2021).\u003c/li\u003e\n \u003cli\u003eKandukuri, S. S., Noor, A., Ranjini, S. S., \u0026amp; Vijayalakshmi, M. A. Purification and characterization of catalase from sprouted black gram (\u003cem\u003eVigna mungo\u003c/em\u003e) seeds. \u003cem\u003eJournal of Chromatography B\u003c/em\u003e, \u003cem\u003e889\u003c/em\u003e, 50-54, (2012).\u003c/li\u003e\n \u003cli\u003eNadaroglu, H., Celebi, N., Demir, N., \u0026amp; Demir, Y. Purification and characterisation of a plant peroxidase from rocket (\u003cem\u003eEruca vesicaria sbsp. Sativa\u003c/em\u003e) (Mill.) (syn. E. sativa) and effects of some chemicals on peroxidase activity in vitro. \u003cem\u003eAfrican Journal of Agricultural Research\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(21), 2520-2528, (2013).\u003c/li\u003e\n \u003cli\u003eAydemir, T. Partial purification and characterization of polyphenol oxidase from artichoke (Cynara scolymus L.) heads. \u003cem\u003eFood chemistry\u003c/em\u003e, \u003cem\u003e87\u003c/em\u003e(1), 59-67 (2004).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Protein, Catalase, Peroxidase, Polyphenol oxidase, kinetic, defence, Adaptation","lastPublishedDoi":"10.21203/rs.3.rs-7436731/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7436731/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eSapindus mukorossi\u003c/em\u003e (soapnut) and \u003cem\u003eAcacia concinna\u003c/em\u003e, belonging to the families Sapindaceae and Leguminosae respectively, are medicinal plants known for their high saponin content as part of their adaptation and defence strategies against environmental stresses. This study investigates and compares the enzymatic antioxidant defence responses of these two saponin rich species, focusing on key enzymes involved in plant stress role in plant adaptation. Enzymatic assays displayed notable differences in the activities of catalase, peroxidase, and polyphenol oxidase; \u003cem\u003eAcacia concinna\u003c/em\u003e possessed higher overall enzymatic activity, while \u003cem\u003eSapindus mukorossi\u003c/em\u003e possessed higher polyphenol oxidase activity reflecting their ecological adaptation and biochemical resilience. The comparative kinetic profiling highlights the enzymatic adaptability of both species under oxidative stress, emphasizing the ecological and biochemical roles of saponins in defence. Linear Discriminant Analysis (LDA), which captured 96.53 percent of the total variation, proved a clear isolation based on enzymatic profiles. Post hoc analysis confirmed statistically significant differences (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) in enzyme activity between the two species. These findings provide insights into the metabolic resilience of saponin-rich plants and contribute to understanding plant defence mechanisms in stressful environments.\u003c/p\u003e","manuscriptTitle":"Enzymatic defence mechanisms in Sapindus mukorossi and Acacia concinna: A Michaelis Menten model approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-21 12:04:45","doi":"10.21203/rs.3.rs-7436731/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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