EVALUATION THE PROPERTIES OF PURIFIED LACCASE EXTRACTED FROM SOME LOCAL PLANTS UNDER THE OPTIMUM CONDITIONS

This study set out to screen 36 common plants which have the greatest level of laccase enzyme activity. It revealed that the enzymatic activity of fenugreek seeds was the highest in comparison with other plants. The optimal enzyme-specific activity was 5340.38 units per milligram protein which were obtained by extracting the enzyme with a sodium phosphate buffer at a concentration of 0.02 M and pH 8.0, at a ratio of 1:40 (weight to volume), and extracting time of 210 minutes. The enzyme yield was 27.6% after extraction and purification by gel filtration using Sephacryl S-3 after 1.01 purification fold. The optimum circumstances for enzymatic activity and stability were found by using 0.1 M sodium acetate as a buffer at pH 5. Also, the maximal activity and stability of purified laccase was obtained at 20 o C for 15 min by using o-tolidine as a substrate. This research sheds light on how to isolate and characterize the laccase enzyme, an important biochemical with numerous biotechnological and technological uses through fenugreek seeds as a source of the laccase.

and Tomato(salamum lvcopersicum). The plant materials were subjected to availability and laccase extraction capacity tests as part of the screening process. To prepare plant specimens, they were cut into small pieces and then dried at room temperature. Chemicals Three buffer solutions, namely sodium acetate (99% C 2 H 3 NaO 2 BDH, England,), phosphatebuffered saline (C l2 H 3 K 2 Na 3 O 8 P 2 , BDH, England), tris-base (C 4 H 11 NO 3 , Hi Media, India), and o-tolidine, HgCl 2 , ZnSO 4 , KCl, FeSO 4 , EDTA, CuSO 4 , CaCO 3 , and cysteine, were bought from Hi-media. The process of extracting and recovering the laccase enzyme: One gram of thirty-six plant was mixed with 40 ml of 0.02 M sodium phosphate buffer solution at pH 7 to extract laccase from the tested plant. The extract solutions were centrifuged at 10000 rpm for 15 minutes. Enzyme activity, protein content, and specific activity were by testing the consequent clear liquid, also called the crude extract (16).

The Laccase activity measurement
To measure the laccase activity; o-tolidine was used as a substrate according to the method described by Al-Assadi (1). Firstly, o-tolidine oxidation detection was measured using a spectrophotometer to screen the clear solution absorbance at 366 nm where: e 366 = 27,600 in units of M -1 cm -1 . Secondly, the laccase protein concentration was determined using the Bradford (8) technique. Then, enzyme activity was calculated by a formula described by Kalral et al.,(19) as follows: Laccase activity (U/ml) =10 6 ×A × V / t × e × v.. (1) Where A is the 366 nm absorbance and V is the total volume (ml) of the combination. v = milliliters of enzyme, t = minutes of incubation. e: the substrate extinction coefficient (e 366 = 27,600 in M-1 cm-1) and is the cuvette's diameter (in centimeters). The quantity of enzyme needed to oxidize 1 μmol of substrate per minute was defined as one unit of enzyme activity. (24). Optimum conditions for laccase extraction: Type of extraction buffer: Three types of buffers were examined to select the best one to be used for further experiments, the experiments were done by mixing the best plant samples obtained from the previous experiments. The experiments were conducted by mixing the plant sample for 15 minutes at room temperature, with 0.02 M sodium acetate (pH 4, 5, and 6), 0.02 M phosphate-buffered saline (pH 7, 7.6, and 8), and 0.02 M tris-base (pH 9, and 10) .The best buffer in terms of enzyme activity, protein content, and specific activity was measured using the same procedures (19,8,17).

Concentration of extraction buffer
The type best buffer was derived from a previous experiment of 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 M was experimented to find the optimum concentration. The best concentration buffer in terms of enzyme activity, protein content, and specific activity was measured using the same procedures (19,8,17).

Extraction time
The purpose of this experiment was to identify the optimal extraction time ranging from (5-240) min for laccase by employing best concentration buffer and pH. The activity of the extracted enzyme was tested, protein content and specific activity were measured. Optimum extraction time was stated in the next experiments.
Extraction ratio: one g of fenugreek seeds was mixed with best concentration buffer and extraction time obtained from previous experiments using the extraction ratio (w:v) of 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, and 1:70 to find the optimal buffer ratio for fenugreek seed enzyme extraction(4). Followed by centrifuging at 10000 rpm for 15 minutes. The measurements of enzymatic activity, amount of protein, and specific activity were conducted (19,8,17). Purification of enzyme with gel filtration chromatography Sephacryl S-300 preparation : A suspension of approximately 20 grams of Sephacryl S-300 was prepared in 500 millilitres of distilled water at a temperature of 90 o C. The suspension was subjected to gentle agitation for a duration of 3 hours to facilitate the swelling of the gel beads. Following this, the mixture was stored overnight in a refrigerator at a temperature of 4 o C, in accordance with the instructions provided by the manufacturer (Pharmacia-Sweden). The gel underwent degassing through the utilisation of a vacuum pump. Subsequently, it was carefully placed within a glass column measuring 31×1.5 cm. The gel was then subjected to equilibration using the identical sodium phosphate buffer that had been employed during the washing and resuspension procedures. Enzyme fractionation using a Sephacryl S-300 column: The enzyme was passed through a Sephacryl S-300 column, and the elution phase was performed with 0.1M sodium phosphate buffer solution, pH 7.6, at a flow rate of 20 ml/h and a fraction volume of 3 ml. At a wavelength of 280 nm, the protein concentrations in each fraction were calculated, after which the enzyme activity of these fractions was measured and the effective fractions were collected, the volume was measured, the activity and protein concentration were estimated, and then the volume was concentrated and distributed among tubes, frozen, and stored for future experiments (4). Characterisation of laccase enzyme Evaluation of the effects of pH and temperature on laccase activity and stability: The effects of reaction pH and temperature were investigated by measuring the activities of laccase at a range of pH and temperatures. The stabilities of the laccase were determined as follows (18). Then enzyme activity and the residual activity (%) were plotted against pH and the temperatures (34,5).

Specificity of laccases
Different substrates, such as toluidine, tannic acid, guaiacol, and catechol, were used to examine their effects on laccase activity. These substrate solutions were prepared by adding 10 mM of each substrate to 15 mL of 0.1 M sodium acetate buffer at pH 5. The best substrates for laccase activity were identified by analyzing the relationship between enzyme activities and substrates. Influence of certain chemicals on laccase activity: On partially pure laccase activity, the effect of several chemical substances, such as HgCl 2 , ZnSO 4 , KCl, FeSO 4 , EDTA, CuSO 4 , CaCO 3 , and cysteine, was investigated. Each compound solution was dissolved in 0.1 M of sodium phosphate buffer at a pH of 5 to produce a solution that was either 5 or 10 mM in concentration. The enzymatic solution was incubated with the metal ion solution at 1:1 (v/v) ratio for 15 minutes at 20 degrees Celsius. After the incubation period, the enzymatic activity was measured and compared to the control. Following that, the percentage of remaining activity was measured (1).

RESULTS AND DISCUSSION Determination of the best plant material:
This study investigated the influence of plant type on laccase enzyme extraction, and the findings showed that there were substantial differences in the enzymatic activity of the 36 plants that were tested. Turnip plant had the highest laccase specific activity, but because it is a seasonal plant, it is only available during the winter months. Fenugreek seeds had the second highest laccase specific activity, measuring 1141.47 U/mg in a 0.02 M sodium phosphate buffer at pH 7. Tomatoes, on the other hand, had the least amount of specific activity ( Fig. 1). Al-Heety (2) suggests that variations in enzymatic activity among plant sources may have origins in both genetics and environmental conditions like temperature, pH, and soil composition. Aziz et al. (5) found that Malva parviflora had a maximum enzyme-specific activity of 420 units/mg proteins

v) Type of extraction buffer
The specific activity of laccase was estimated after extraction using different buffers, and the findings are presented in Figure 2. Accordingly, the sodium phosphate buffer with a concentration of 0.02 M and a pH of 8.0 was the ideal extraction buffer since it had a specific activity of 1293.31 U per mg of protein. The specific activity of other buffers with various pH levels was quite low. According to Gawlik-Dziki and colleagues (15), most plant species exhibit the highest levels of enzyme activity at or very close to pH 7. The presence of H+ and/or OH-ions in an enzyme creates competition with hydrogen and ionic bonds, enzymatic denaturation occurs as a result Tortora et al., (38). A buffer solution that can preserve protein stability throughout the entirety of the procedure needs to be chosen. This is necessary to keep the extraction process on schedule and effective, as well as to avoid the necessity of switching buffer solutions in the middle of the process (20). According to Al-Sa'ady and Hilal (3), the optimal buffer for the laccase extraction from M. parviflora was a solution of 0.2 M sodium acetate at a pH of 6. This buffer offers a specific activity level of 1.8 U/mg and was found to be the perfect choice. According to Niladevi and Prema (29), the value pH for Streptomycetes sp laccase extraction ranges between 7 and 7.5.

Figure 2. Effect of the type of buffers on laccase extraction from fenugreek seeds at 25 °C for 15 min The concentration of extraction buffer
Effect of the concentration of extraction buffer on the activity of laccase extracted from fenugreek seeds was studied. Result illustrated in figure (3), a solution of 0.02 M sodium phosphate buffer at a pH of 8 is the optimal choice for the process of extracting laccase from fenugreek seeds. This concentration produced the maximum specific activity, which was measured at 1295.29 U/mg protein.
Indicating that the concentration of buffer can have a substantial effect on the enzymatic activity, the low concentration of 0.01 M resulted in a low specific activity of 322.96 U/mg protein. The high concentration of buffer has a negative impact on the activity of laccase. This is likely due to the presence of numerous ionic groups that make the job of enzymatic activation more difficult. According to Al-Heety (2), it is essential to pick the right buffer concentration to optimize the extraction process and preserve the enzymatic activity.

Figure 3. Influence of buffer concentration on extract laccase from fenugreek seeds at 25 °C for 15 min Extraction time
The purpose of this experiment was to identify the optimal extraction time for laccase by employing a sodium phosphate buffer with a pH of 8.0. The specific activity of the extracted enzyme was tested, and the results are depicted in Figure 4, which displays the outcomes of 10 distinct extraction times ranging from 5 to 240 minutes each. After 210 minutes of extraction, the findings showed that a specific activity of 5,340.38 U/mg protein was obtained, which was the maximum possible value. It is important to note that the optimal extraction time may differ depending on the source of the enzyme and the materials that are present in that source. These materials might interfere with the enzyme, and contaminants can impair the stability of the protein extract regarding its ability to resist decomposition. In order to maximize the effectiveness of the enzyme extraction process and achieve the maximum possible level of specific activity, it is necessary to identify the optimal extraction duration for each unique source of enzyme. This finding is consistent with previous studies that highlight the importance of optimising extraction conditions to obtain high-quality enzyme extracts (30). Extraction ratio Different buffer ratios were using to determine the optimum for maximum laccase activity under optimum conditions examined previously. The extract ratio of 1:40 ratio had the highest specific activity, measuring 5128.09 U/mg protein; specific activities were also detected at 3781.45, 1768.59, 1084.11, 914.05, 902.88, and 876.76 U/mg protein with the other ratios (Fig. 5). The extraction ratio varies depending on the enzyme's composition and concentration. As the concentration of the extraction solution rises, the rate at which the enzyme-reactant complex is formed slows down, reducing the specific activity of the enzyme (21). The proportion of the herb used in the extract, known as the native extract ratio, can vary. As a result, the equivalent dry weight of the plant can vary depending on how much of the herb is used in each preparation. According to Predescu et al. (31), a low native extract ratio is associated with a high yield of extractable material from the herbal source. Fenugreek seeds were homogenized with sodium phosphate buffer (0.02 M, pH 8.0) at a ratio of 1:40 (w:v) for 210 min, and the laccase enzyme was subsequently extracted by cooling centrifugation at 10,000 rpm. The crude laccase was subjected to purification through gel filtration using Sephacryl S-300 in order to remove unwanted proteins and other components.

Gel filtration chromatography
To begin, a 0.1 M sodium phosphate buffer at pH 7.6-equilibrated Sephacryl S -300 gel column (31 cm × 1.5 cm) was used to filter crude enzyme extract. The analysis highlights a pair of protein crests in proportions eluted from this filtration, with a further additional high point indicating the presence of laccase (Fig. 6), so the appropriate fraction of the filtrate can be collected. Table 1 demonstrates that there was only one protein peak and one laccase activity peak in the fractions eluted from the column. Active laccase was present in all of fractions 12-31, with a maximal specific activity of 5228.9 U/mg protein with 1.01-fold of purification and a yield of 27.6% overall. Purification using Sephadex G-100, the crude laccase recovered from Trametes polyzona WRF03 (TpL) revealed a single peak at 280 nm in UV spectra, confirming the presence of a single, distinctive feature. With a purity level 13 times higher than 2%, post-gel filtration laccase had a protein-specific activity of 21525 U/mg when measured with ABTS (13). Laccase from M. parviflora was purified using Sephadex G-150 to obtain a protein with a specific activity of 47830 U/mg and a purity of 94.4% (5).

6) at a flow rate of 20 mL/h, with each fraction collected in 3 mL Characterization of partial purified laccase
The effects of different parameters on the purified laccase are determined pH's influence on laccase activity The effect of the pH of three different buffers on the activity of partially purified laccase were studied. A o-tolidine was used as a substrate with varying pHs, including 0.2 M sodium acetate buffer (pH 4, 5, and 6), 0.2 M phosphate buffer (pH 7 and 8), and 0.2 M of tris-base buffer (pH 9 and 10). The optimum pH for laccase activity was discovered by estimating the activity and then plotting the relationship between enzymatic activities and pH values. According to the findings, an enzyme's activity is at its highest when the pH is 5, this results in enzyme activity reached 68322.98 U/mL. The decline in activity was also seen to be noticeably more pronounced in the alkaline medium as compared to the acidic medium. The pH may affect the organization of both the enzyme and the substrate, The ionization of groups within the active site of an enzyme, as well as the ionization of groups within the substrate (11). This is one of the many ways that pH can affect enzymatic activity. The observed phenomenon of increased substrate oxidation at higher pH levels may be attributed to the significant difference in redox potential between the phenolic substrate and the T1 copper. However, when hydroxide anions (OH -) bind to the T2/T3 coppers, laccase activity is inhibited because this stops electrons from readily passing across the T1 and T2/T3 centers. Consequently, laccase activity is reduced (40). The pH of enzyme stability was studied because it is an important criterion to determine the optimum conditions for purification and storage of the enzyme. At varying pH levels (ranging from 4 to 10), a mixture of partially purified enzymes and buffers was combined in equal amounts (1:1 ratio). Thereafter 15 minutes in a water bath maintained at 37 o C. The tubes were placed straight into an ice bath for further examination. After measuring the enzymatic activity, the remaining enzymatic activity was calculated for each pH value and plotted to determine the optimum pH for laccase stability. This was done to determine the best pH. According to the findings presented in Figure (8), the most favourable conditions for the stability of laccase are reached at pH levels ranging from 4 to 7. While enzyme activity stayed at 100% when the pH was 5, it remained at around 89.81% when the pH was 4. In addition, it preserved around 86.56 percent and 78.72 percent of its initial activity when the pH was 6 and 7, respectively. A decline in activity was observed on both sides of the optimal pH values, with residual activities above 71.83% at pH 8, and 63.44% and 54.50% at pH 9 and 10, respectively. It was shown that the enzymatic activity decreased as the pH level became more alkaline. It is possible that the influence of pH stability on the structure of the enzyme is responsible for the general drop-in activity that is observed at pH levels that are outside of the optimum range. The secondary and tertiary structures of the enzyme molecule are altered because of this, which may result in the enzyme becoming denatured or in alterations to the ionic state of the active site. According to Segel (35), enzymes lose their activity when exposed to buffer solutions with pH values that fall outside of the optimum range.

Effect of temperature on laccase activity
Temperature is an important factor which affects enzyme activity. The favourable temperature for laccase activity may differ with different laccase sources. The activity of partially purified laccase was determined at temperatures of 20, 37, 45, 50, 55, and 60 o C. The optimal temperature for laccase activity was then determined by analyzing the correlation between enzyme activities and temperature. The optimal temperature for laccase activity was determined by subjecting the purified enzyme and substrate combination to various temperatures ranging from 20 °C to 60 °C, with an incubation period of 15 minutes. The enzyme's activity was found to be significantly higher at 20 °C (as shown in Figure 9). At 20 °C, laccase activity was at its highest (65607.14 U/mL), but it dropped to its lowest (17698.94 U/mL) at 60 °C. Due to the fact that within a given temperature range, the rate of enzymatic interaction accelerates as a result of enhanced energy kinetics and collisions between enzyme molecules and substrate, laccase activity was also seen to drop below 40 °C. Muro et al. (28) noticed that enzymes lose their three-dimensional structure and enzymatic function when exposed to temperatures above certain thresholds, a process known as denaturation.These findings corroborated Kim et al. (22) works, who employed o-tolidine as a substrate to demonstrate that the optimal temperature for the activity of enzyme isolated from Phlebia termellen is 20 °C. Cladophora sp. laccases remained active at 35 °C (2). The partially purified laccase enzyme was subjected to a series of temperature treatments (20,37,45,50,55, and 60°C) for a duration of 15 minutes. Subsequently, the enzyme was promptly transferred to an ice bath. Following that, the enzymatic activity was evaluated at the temperature that maximizes enzyme activity. The remaining activity percentage was estimated. Figure (10) shows that incubation of laccase at temperatures higher than the optimal for stability (20 °C) resulted in a slight decrease in the enzyme's remaining activity, with maximum remaining activity being achieved at 20 °C. The enzyme was still 94% as active at 60 °C as it was at room temperature. At lower temperatures, most enzymes are more stable, which is why they need to kept in the fridge or freezer. The fact that laccase activity decreases at 60 °C demonstrates its sensitivity to high temperatures, which may alter the protein's three-dimensional structure by damaging Rgroups of amino acids. Denaturing proteins in this way causes them to stop working as enzymes (9).

Effect of substrates on Laccase specificity
In order to investigate the specificity of purified laccase extracted from fenugreek seeds toward different substrates. Different substrates were added to the purified enzyme and the reaction mixture was incubated, then the activity determined. As can be seen in Fig.  (11), when o-tolidine was used as a substrate, the enzymatic activity was significantly higher than when other substrates were used. Activity of laccase was as high as 7686.34 U/mL but dropped when other substrates were used. Furthermore, laccase activity reached 34161.49, 11956.52, and 3416.15 U/mL when using tannic acid, guaiacol, and catechol, respectively; the findings of this study indicate that the laccase obtained from fenugreek seeds exhibits enhanced oxidation capabilities when o-tolidine is employed as the preferred substrate, surpassing its performance with other phenolic compounds. Orthotolidine was shown to be the phenolic molecule most amenable to oxidation by the laccase enzyme isolated from Cladophora sp. (2). Figure 11. Illustrates the impact of various substrates on fenugreek seeds purified laccase activity at a temperature of 20 °C and pH 5 Influence of certain chemicals on laccase activity: Heavy metals normally found in the environment, might affect the activity or stability of enzymes. The effect of some heavy metals and chemicals agent on the activity of the purified laccase was examined. According to the findings of Fig. 12, the concentration of KCl that produced the greatest increase in laccase activity was between 5 and 10 mM. According Cliff et al. (10), metals can be assimilated as a component of enzyme cofactors, which boost the activity of the enzymatic reaction, and they can also be adsorbed to the surfaces of cells and precipitated as a result of the metabolic process. In addition, the enzyme was activated by CuSO 4 and FeSO 4 at concentrations of 5 and 10 mM, respectively. Telke et al., (36) elucidated that CuSO 4 has a role in enhancing the rate of o-tolidine oxidation. Additionally, it was stated that the stimulation of laccase activity by CuSO4 could potentially be attributed to the binding of copper ions to the type-2 copper binding sites. Due to its natural occurrence as a biogenic metal in the environment, the concentration of copper can significantly influence the activity of enzymes, affecting their ability to perform their functions and maintain their stability. The effect that metal ions have on the activity of laccase might change depending on where the enzyme came from, often known as its source. At doses of 5 and 10 mM, it was noted that the presence of HgCl 2 inhibited enzymatic activity to the point where it reached levels that were lower than the activity that was being controlled. These outcomes are consistent with the majority of studies that investigated the influence of these compounds on laccase activity. According to the study conducted by Farnet et al (14) the inhibition of laccase activity was attributed to the effect of metal ions, particularly chloride ions. The enzyme was inhibited when HgCl 2 was present, which demonstrated the presence of sulfhydryl (SH) groups inside the active region of the enzyme, which were oxidized when HgCl 2 was present. According to Maher and Cordes (26), the presence of HgCl 2 in the solution that is being used for the processing of the substrate might lead to the development of a complex with the enzyme. This complex would prevent the enzyme from attaching to the substrate, which would slow down the process of product creation. During incubation with a ZnSO 4 solution, there was an increase in the enzyme's enzymatic activity at both 5 and 10 mM, with the respective increases being 150.34% and 106.50%. The increase in laccase activity was seen with CaCO 3 at concentrations of 5 and 10 millimolar (109.80% and 115.86%, respectively). These findings are consistent with those that were published by Bao et al (6) and Ma et al. (25), who found that the enzymatic activity was significantly improved by 1 and 10 mM CaCO 3 (12% and 35%, respectively). Ca +2 has the capacity to promote laccase activity at concentrations as low as 1 mM, even when the concentration is not very high. Ca +2 ions are necessary for the catalytic activity of laccase due to this reason; however, they are not necessary for the structural stability of laccase. EDTA and cysteine, when present at doses of 5 and 10 mM, appear to have the opposite effect on the activity of laccase, which have been previously reported as an efficient laccase inhibitor (1,2). This stands in opposition to the other attributes of laccase. The resistance to EDTA as a chelating agent can be attributed to its limited ability to access the structural copper atoms located in the active site, which are necessary for the catalytic activity of the enzyme (33). As for cysteine, which may suggest no strong reduction of the disulphide bridges between domains 1 and 2, which stabilizes the structure of laccase (7).