2.1. Chemicals and Materials

Sodium-potassium Tartrate, casein, albumin, and cupric sulfate pentahydrate (Sigma-Aldrich, Germany). Sodium carbonate and egg albumin (BDH .GPR. England). Phenol reagents (Folin–Ciocalteu) (s.d. fine-CHEM Ltd). Sodium phosphate-3-hydrate and sodium acetate-3-hydrate (DAB, PH.EUR, B.P., PH. Franc). Acetic acid glacial (NenTech Ltd., UK). Trichloroacetic acid (TEDIA Company, Inc., USA).

Mentha piperita L. (Mentha) and Thymus capitatus L. (Thymus) freshly collected from local markets (once) in September 2014–2015. The plants’ aerial parts (leaves) thoroughly washed with distilled water and refrigerated at 4oC until used. The research was done in the biochemistry lab at the Department of Biological Sciences, Mutah University.

2.2. Crude Plant Extract

Forty grams of plant leaves were ground by an electric homogenizer. The suspensions were finely powdered and mixed with phosphate-buffered (0.1 M, pH 7.0). Then, the homogenized mixture was filtered and centrifuged for 10–15 min. The collected supernatant was considered as the crude plant extract (enzyme solution) [1].

2.3. Protein Measurement

Protein concentration determined by the Lowery colorimetric method [25]. The bovine serum albumin was the standard used. The standard curve used to determine the protein amount in the extracts (mg/ml).

2.4. Protease Assay

The protease activity is determined by Fahmy method [17]. Briefly, to an Eppendorf tube, 50 ml of the crude plant extract (enzyme solution) was added to 650 ml of 50 mM phosphate-buffered (pH 6.5) and 100 ml of egg albumin (as a substrate) at 37°C for 1 h. Then, 200 ul of 10% trichloroacetic acid was added to stop the reaction. After centrifugation, the absorbance of the supernatant measured at wavelength 360 nm using UV-Vis spectrophotometer (Biotech Engineering Management Co. LTD, UK). The activity of protease was defined as the amount of enzyme that hydrolyzes 1 mmol of amino acids (in terms of tyrosine) from egg albumin per minute under the standard assay conditions.

2.5. Influence of pH and Temperature on Enzyme Activity

The acetate buffer (50 mM, pH 2.0–5.0) and phosphate-buffered (50 mM, pH 6.0–10.0) were used to determine the effects of pH on the enzyme activity [3]. The initial velocity data obtained at different pH values used to indicate and determine the possible ionizable groups in the active site of the protease enzyme by using Dixon–Webb’s plot [26]. The temperature range 25–60°C was used to perform the effects of temperature on the enzyme activity [3].

2.6. Substrate Specificity and Kinetic Studies

Substrate specificity was determined as a relative proteolytic activity of crude plant extracts of proteases by the Patil method using egg albumin as a substrate with minor modifications. Kinetic experiments performed using varying concentrations of substrate (1–3%) and Line weaver–Burk plot used to determine the K_m, and the V_max [27].

2.7. Statistical Analysis

The assays were performed in triplicate and expressed as mean ± standard deviation (SD). The statistical analysis was performed by t-test, if P ? 0.05 was statistically significant.

3.1. Estimation of Protein Concentration

The protein concentrations in crude plant extract estimated by the Lowry method. Table 1 shows the protein concentration in the crude extracts of the selected plants. From the standard curve, the protein concentration in the extracts calculated. The highest protein concentration found to be in the Mentha (58.16 mg/ml) and low concentration in Thymus (5.67 mg/ml), relatively.

Table 1: Protein concentration (μg/ml) in the crude plant extracts of the selected plants. Data presented are Means±SD of n=3 experiments

3.2. Estimation of the Optimum pH and the Ionizable Groups

For Thymus, the profile of pH relative activity ranged between pH 2.0 and 3.5 with maximum relative activity 65% and at pH 6.0–8.0 with maximum relative activity 100% [Figure 1]. Therefore, Thymus has two optimum pH at 3.0 and 6.5. On the other hand, Mentha has two optimum pH at 3.0 and 9.0 with relative activity 100% and 70%, respectively [Figure 1]. In general, the presence of two optimum pH may be due to the presence of isoenzymes for the particular protease.

Figure 1: The relative activity (%) of the pH profile of the protease enzymes in crude plant extracts of Thymus and Mentha. Data presented are means±SD of n = 3 experiments [Click here to view]

In the literature, it was reported that the optimum pH for most plants is different and it was ranged from 3.0 to 10.0. For example, Ali et al. [28] reported that Carum copticum has two peaks at pH 3.0 and pH 7.0 while for Allium cepa has one optimum pH at 10.0 (alkaline). In comparison with fungal proteases, the majority of the pH optimum ranged between 8.0 and 12.0, while for bacteria are varied from one strain to another and ranged between 3.0 and 11.0 [29].

Dixon–Webb’s plot used to indicate and determine the ionization groups in the active site of the protease enzymes. According to the Dixon and Webb’s “protein conformation can be maintained within 4-5 pH units but activity different,” it means that the ionizable groups of amino acids in the active site are affected by pH. The analysis of the Dixon–Webb’s plot of the Thymus and Mentha is shown in Figures 2 and 3, respectively, these figures illustrate the presence of aspartic acid and cysteine amino acids in both of the selected plants, which indicate that proteases in the crude plant extracts of these plants may belong to the acidic and neutral proteases.

Figure 2: Dixon–Webb’s plot for the determination of possible ionizable groups in the active site of proteases of Thymus using egg albumin as a substrate. Data presented are means ± SD of n = 3 experiments [Click here to view]

Figure 3: Dixon–Webb’s plot for the determination of possible ionizable groups in the active site of proteases of Mentha using egg albumin as a substrate. Data presented are means ± SD of n = 3 experiments [Click here to view]

In both Thymus and Mentha, the ionizable groups found to exhibit two types. One type with pKa₁ at pH 6.7 and pKa₂ at pH 7.2 which suggests the presence of the amino acid cysteine in the active site of the enzyme and therefore maybe belong to the neutral proteases group (Cysteine protease), while the other ionizable groups have pKa₁ at pH 3.2, and therefore maybe belong to the acidic proteases group (Aspartic protease) [Figures 2 and 3]. The results of pKas, suggest the presence of aspartic acid and cysteine amino acids in and around the active sites of these protease enzymes [30,31].

Aspartic proteases (EC 3.4.23) are that proteolytic enzymes have two aspartic acids in their active sites. On the other hand, the catalytic mechanisms of the cysteine proteases, it involves the nucleophilic cysteine sulfur group in the catalytic dyad. In general, the cysteine proteases such as Actinidain, Bromelain, Calpains, Caspases, Papain, and others are found in fruits, leaves of different plant families.

3.3. Estimation of Optimum Temperature

The optimum temperature of an enzyme is considered a very important factor especially for those proteases used in the industry. In general, most industrial enzymes require a higher temperature than physiological enzymes.

Figure 4 shows that maximum relative activity (100%) for proteases of Thymus at temperature 40°C. The activity profile of the temperature for Mentha was different. The range of the activity from 25 to 60°C with the lowest relative activity 60% at temperature 45°C and high activity at temperature 35°C ( 100%) and relative activity 90% at temperature 50°C [Figure 4]. This thermal stability of proteases may be due to the presence of some metal ions needed for their biological activities [4]. The activity profile for elevation temperature above 50°C for all plants shows a sharp decrease in the activity. The explanation for these results depends on the protein nature of enzymes that are the elevation of temperature above 60°C leads to protein denaturation.

Figure 4: The relative activity (%) of the temperature profile of the protease enzymes in crude plant extracts of Thymus and Mentha. Data presented are means±SD of n = 3 experiments [Click here to view]

3.4. Determination of K_m and V_max

In addition to pH and temperature, substrate concentrations considered as one of the main factors for determining the activity of the enzymes. In addition, it can be used for determining the enzyme kinetics (K_m and V_max) to illustrate the enzyme affinity and specificity. In the experiments, different concentrations of egg albumin (0.5–3.0%) were used. It was found that V_max was reached at a concentration of 3% for the egg albumin substrate.

Line weaver-Burk plots were used to calculate the K_m and V_max of proteases enzymes. Figure 5 illustrates the plots of K_m and V_max for the proteases of the Thymus and Mentha and all the results summarized in Table 2. Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max) of the protease enzymes were determined using the above substrate.

Figure 5: Determination of Km and Vmax values for protease enzyme in the crude plant extracts of the Thymus and Mentha using egg albumin as a substrate. Data presented are means ± SD of n = 3 experiments [Click here to view]

Table 2: Comparison between the V_max, K_m, and the ratio of V_max/ K_m for the crude plant extracts (proteases) of the selected plants in means of egg albumin as substrates

Studying the kinetics of an enzyme considered a very important value in understanding the affinity and conditions suitable to obtain optimum activity, especially, if these enzymes used for industrial and biochemical applications. Proteases are one of the most groups of enzymes that play a role in many fields of industry.

According to the obtained data from Line weaver-Burk plots, substrate specificity (V_max/K_m) was calculated. Results in Table 2 showed different affinities toward egg albumin as a substrate. K_m value is a measure of the affinity of the proteases for the substrate, the smaller the value the higher the affinity of the enzyme for that substrate while Vmax shows the catalytic efficiency, the higher the V_max the higher the efficiency.

In general, the best substrate for any enzyme depends on the strong binding of the substrate to the enzyme (low K_m) and high proteolytic activity (high V_max). It was found that the egg albumin has more affinity to toward Mentha proteases (Km 1.7 mg/ml) than Thymus proteases (K_m 3.33 mg/ml), while Thymus proteases have higher catalytic efficiency (V_max 2.94 mmol/min) than Mentha proteases (V_max 1.3 mmol/min). By considering the ratio (V_max/K_m) [Table 2], the observed results indicated that egg albumin was a good substrate for proteases of Mentha (0.764) and Thymus (0.882).