3.1 The effect of pH on the Chlase activity in the mulberry leaves
The effect of pH on the Chlase activity in the mulberry leaves was estimated (Fig. 1). The results indicated that the Chlase activity was obviously pH-dependent with complete inactivation at pH 2.0. The Chlase activity rose slightly at a pH level below 3.5, followed by a substantial increase from pH 3.5 to 6.5, reaching a maximum activity value at pH 7.5. This activity started to decline at a pH value above 8.0. These results showed that the Chlase activity was more effectively inhibited in acidic pH conditions than in an alkaline environment, ranging between 0.402 U/mg and 0.688 U/mg. The increase in Chlase activity from pH 3.5 could be attributed to a decrease in the H+ ion concentration and more significant enzymatic active center availability (Muhammad et al., 2021). Chlase denaturation occurs at pH levels below 4.0 or above 8.0, leading to enzymatic inactivation (Li et al., 2019; Do et al., 2009). In addition, a strongly acidic or alkaline environment might affect the dissociation of the relevant groups on the substrate (Fernández and Kelly, 2016). Previous studies showed that the endogenous enzymes in vegetables and fruits were often inactivated at pH values below 4.0, providing a method for controlling enzymatic degradation and hydrolytic reactions (Fernández and Kelly, 2016; Ge et al., 2022; Chattopadhyay et al., 2023).
The results of this study were consistent with the findings for green tea leaves (Okazawa et al., 2006; Sytykiewicz et al., 2013; Ozkan and Ersus Bilek, 2015). Okazawa et al.(2006) reported that a pH level of 7.5 was optimal for Chlase extraction from green tea leaves. Similarly, Sytykiewicz et al.(2013) found that the optimum Chlase pH in wheat seedlings ranged from 7.0 to 8.0. Ozkan and Ersus Bilek (2015) reported that the Chlase activity in spinach was highest at pH 7.0. The differences in the optimum pH values for Chlase activity could be attributed to variations in plant sources, extraction methods, enzyme purity, buffers, and substrates (Fernández and Kelly, 2016).
3.2 TP inactivation kinetics of mulberry Chlase in distilled water
No significant loss in Chlase activity loss was evident in the mulberry leaves when heated at temperatures below 70 ℃. Consequently, the thermodynamic analysis of the thermal Chlase inactivation occurred at temperatures ranging from 75 ℃ to 100 ℃. Figure 2a shows the inactivation results of mulberry Chlase in distilled water. The Chlase inactivation rose as the temperature and treatment time increased. The RA of the Chlase declined rapidly within the initial 5 min, while the inactivation rate decreased significantly after prolonged thermal treatment. Treatment at 75 ℃ and 80 ℃ for 28 min reduced the Chlase RA by 34.31% and 28.45%, respectively, while temperatures higher than 80 ℃ distinctly accelerated Chlase denaturation. After 18 min of heating at 85 ℃, the Chlase activity decreased by 20.98%, while over 86% and 90% of the Chlase were inactivated when treated at 90 ℃ and 95 ℃ for 16 min and 10 min, respectively. Increasing the temperature to 100 ℃ visibly decreased the thermal stability of the Chlase, while the RA was lower than 1% after an 8-min treatment. The results indicated the presence of at least two Chlase isozymes in the mulberry leaves, which could be divided into TP-sensible and TP-resistant fractions. Previous studies purified and characterized Chlase isozymes in various plant and algal species, including green tea leaves (Li et al., 2011), citrus fruit peels (Rodrigo et al., 2013), Phaeodactylum tricornutum (Su et al., 2023), and Chenopodium album leaves (Shioi et al., 1991), with molecular mass values ranging from 27 kDa to 65 kDa. The isozyme molecular structure differences could cause thermal stability variation. Studies have reported that spinach-derived Chlase contained sensible and resistant fractions after various treatments. Huang et al. (2010) revealed that the Chlase extracted from spinach was effectively inactivated by thermal (55 ℃ and 75 ℃/0 ~ 20 min) and high-pressure carbon dioxide (10 ~ 30 MPa/0 ~ 45 min) treatment, while a minimal RA of 5.53% was detected in samples treated at 30 MPa for 45 min. Similarly, Wang et al. (2012) found that the Chlase in spinach pulp exposed to high hydrostatic pressure treatment (200 ~ 600 MPa/5 ~ 25 min/25 ℃) was partially inactivated by 22.35%~56.77%, and the residual enzymes were pressure stable. However, several studies produced contrary findings, indicating that treatment at 48 ℃ for 3 h activated the Chlase in broccoli (Lemoine et al., 2009), while hot steam at 100 ℃ for 1 min caused Chlase activation in spinach (Wang et al., 2012). The differences in the Chlase susceptibility to high temperatures, high pressures, and high-pressure carbon dioxide may be ascribed to their plant sources. Previous research proposed that the heat stability of endogenous plant enzymes depended on the cultivars, pH, isoenzyme forms, total solids level, and extraction methods while also largely relying on their origin (Martinez et al., 2018).
The relationship between the Chlase RA and the TP treatment time at 75 ℃-100 ℃ was described reasonably well using two-fraction kinetics models, with correlation coefficients (R2) ranging from 0.966 to 0.997 (Table 1). Huang et al. (2010) revealed that the inactivation of crude Chlase extracts in spinach via high-pressure carbon dioxide (55 ℃/0 ~ 45 min/10 ~ 30 MPa) displayed two-fraction kinetics linear behavior (R2 = 0.962 ~ 0.997). First, the RA decreased rapidly to 30% within the initial 10 min of treatment, while only 18% of the Chlase was inactivated during the next 35 min. However, the inactivation kinetics of the Chlase in green tea leaves reportedly followed a simple linear behavior at 30 ℃~60 ℃, while it was described as a first-order model at 70 ℃ and 80 ℃ (Li et al., 2011).
Table 1 shows the kinetic parameters estimated via linear regression analysis. The proportion of TP-sensible Chlase increased gradually from 0.394 to 0.745 as the temperature rose from 75 ℃ to 100 ℃. The corresponding stable proportions decreased from 0.606 to 0.255, possibly due to the transformation and conversion of the Chlase isozyme structure caused by thermal treatment. The inactivation rate constants of the TP-sensible Chlase varied from 0.581 min− 1 at 75 ℃ to 1.374 min− 1 at 100 ℃, with the corresponding D-values changing from 4.79 min to 0.93 min, respectively. The kR and DR kinetic parameters (Table 1) indicated that temperature promoted the denaturation of resistant Chlase. The estimated ZS and ZR values of the Chlase in the mulberry leaves were 36.40 and 21.03 ℃ R2 = 0.961 and 0.837 (Fig. 3a), while the thermal Ea of the labile and stable Chlase was 68.02 kJ/mol and 110.32 kJ/mol, respectively.
3.3 The effect of pH on the TP inactivation kinetics of the Chlase in mulberry leaves
A pH level lower than 4 and higher than 8 effectively inhibits Chlase activity, while strongly acidic and alkaline environments are not conducive to Chl retention in plants (Li et al., 2011). Therefore, medium pH levels (5.7, 6.8, and 8) were used to investigate the Chlase thermodynamics.
The inactivation kinetic curve slopes (Fig. 2b-d) and the inactivation parameters (Table 1) showed that the sensible and resistant Chlase proportions in the mulberry leaves changed with the treatment buffer pH. No significant differences were evident between the AS values of the Chlase in the samples treated with distilled water and phosphate buffer at a pH level of 6.8 and a thermal temperature between 75 ℃ and 80 ℃. However, when the temperature exceeded 80 ℃, the sensible Chlase proportion in the mulberry leaves in the phosphate buffer (pH 6.8) decreased compared with those in distilled water at the same treatment temperature. This indicated that phosphate buffer prevented stable Chlase conversion to labile enzymes during high thermal treatment. At a temperature between 75 ℃ and 100 ℃, no obvious changes were evident in the inactivation rate constants of the TP-sensible Chlase (kS) at pH 6.8 in the phosphate buffer compared to those in distilled water, while the kR notably decreased by 20.45%~322.85% and the DR values increased by 6.45%~163.92%. The results implied that phosphate buffer enhanced the heat resistance of the Chlase in mulberry leaves, likely due to the maintained pH in the phosphate buffer system, while a distinctly lower pH was evident in the distilled water. The pH decreased from 6.8 to 6.6, 6.5, 6.5, and 6.4 after TP treatment at 85℃ for 18 min, 90 ℃ for 16 min, 95 ℃ for 10 min, and 100 ℃ for 7 min in distilled water, respectively. Previous studies reported a similar pH decline after the TP treatment of fruit and vegetable products (Medina-Meza et al., 2015; Huang et al., 2010; Wang et al., 2013a). Wang et al.(2013a) indicated that TP treatment (100 ℃ steam for 1 min) decreased the pH of spinach pulp from 5.89 to 5.76, followed by a continuous decline to 5.34 after 34 d of storage at 4 ℃, possibly due to the destruction of organelles and the release of their cellular content, such as oxalic acid (Zhao et al., 2014). However, strong water ionization at high temperatures may also decrease the pH. The sodium ions in the phosphate buffer may also affect the activity and thermal inactivation kinetics of Chlase. Liu et al.(2010) showed that sodium ion concentrations lower than 0.6 mg/mL activated protease activity, possibly due to stable cluster ion formation between the metal ions and enzyme nucleobases. The estimated ZS and ZR values of the Chlase in the mulberry leaves treated with phosphate buffer at a pH of 6.8 were 38.71 and 22.98 ℃ (R2 = 0.969 and 0.981 (Fig. 3c)), while the corresponding Ea for the thermal inactivation of the sensible and resistant Chlase were 64.26 kJ/mol and 120.27 kJ/mol, respectively.
The Chlase treated in phosphate buffer at pH 5.7, and a temperature ranging from 75 ℃ to 95 ℃ exhibited the lowest RA compared with samples treated in distilled water or phosphate buffer at pH 6.8 and 8.0 at the same temperature and time (Fig. 2b and Table 1). This indicated that the Chlase became increasingly susceptible to the treatment temperature at low pH levels. The kS and kR values of the Chlase treated at pH 5.7 changed from 0.726 min− 1 to 1.819 min− 1 and 0.0253 min− 1 to 0.269 min− 1, while the corresponding DS and DR decreased from 4.55 min to 0.99 min and 91.01 min to 8.59 min, respectively. Specifically, the inactivation kinetics of the Chlase treated in phosphate buffer at pH 5.7 and 100 ℃ was described reasonably well by first-order kinetics (Fig. 2c) with an R2 of 0.962 and an inactivation rate constant of 1.426 min− 1 with the corresponding D value of 1.62 min, suggesting that the stable Chlase disappeared in high-temperature, acidic environments. The higher H+ ion concentration and lower enzyme active center availability caused Chlase denaturation and reduced thermal stability (Liu et al., 2013). It may also be attributed to a decrease in the number of non-covalent bonds in the Chlase molecule, leading to dramatic changes in the secondary and tertiary structures of the Chlase. Liu et al.(2013) found that 5 mM, 10 mM, and 15 mM citric acid concentrations changed the secondary structure of polyphenol oxidase (PPO) and decreased its thermodynamic parameter values. These results were consistent with the findings of Cui (2023), who reported that higher acidity increased PPO inactivation. de Oliveira Carvalho and Orlanda (2017) noted that the heat stability of crude PPO extract from Buriti fruit was inhibited at a pH below 5.0. The thermodynamic Z and Ea values were significantly influenced by pH and declined as the pH decreased. The ZS and ZR of the Chlase treated in phosphate buffer at pH 5.7 decreased to 20.26 ℃ and 35.05 ℃ (R2 = 0.835 & 0.804), while the EaS and EaR declined to 50.06 kJ/mol and 117.38 kJ/mol, respectively. The lower Ea implied that a smaller temperature change was necessary for enzymatic inactivation. Therefore, the Chlase in acidic systems was more susceptible to temperature elevation than in neutral environments.
Increasing the pH to 8.0 in phosphate buffer decreased Chlase inactivation as compared with the pH 5.7 and 6.8 samples at the same treated temperature and time. The kS and kR values of the Chlase in phosphate buffer at pH 8.0 ranged from 0.581 min− 1 to 1.174 min− 1 and 0.0208 min− 1 to 0.422 min− 1, respectively, while the corresponding D values increased accordingly. Therefore, it was inferred that phosphate buffer at pH 8.0 prevented Chlase inactivation during thermal treatment. The estimated ZS and ZR values of the Chlase treated in phosphate buffer at pH 8.0 were 36.11 ℃ and 22.68 ℃ (R2 = 0.843 and 0.870 (Fig. 3d)), while the Ea of the sensible and resistant Chlase was 69.64 kJ/mol and 108.34 kJ/mol (R2 = 0.824 and 0.985), respectively. The improved temperature stability of the Chlase in weakly alkaline conditions likely contributed to the protection of pivotal groups on the active enzyme center (Liu et al., 2010).
Verification experiments were performed to evaluate the fitted inactivation kinetics model. Figure 4 and Table 1 show the correlation between the experimental and model RA of the Chlase in mulberry leaves. A good fit constitutes low SS and RMSE values and a high regression coefficient (R2) value. In the present study, the Af, Bf, SS, and RSME values of the verification experiments in distilled water (pH 6.8) were 1.011, 1.021, 0.0474, and 0.0131 with a high coefficient (R2 = 0.985), implying a good fit between the observed and predicted two-fraction kinetics values. Similarly, the two-fraction kinetics also presented a good fit for the Chlase inactivation after treatment in phosphate buffer at pH 5.7, 6.8, and 8.0.
3.4 The effect of calcium chloride on the Chlase activity in mulberry leaves
As shown in Fig. 5, calcium chloride affected the Chlase activity in mulberry leaves, while the changes were concentration-dependent. Although the Chlase activity changes were negligible after adding 0.1 g/kg and 0.2 g/kg of calcium chloride, it increased by 2.35% after 0.3 g/kg calcium chloride treatment. The Chlase activity in mulberry leaves increased continuously as the calcium concentration rose, with the highest activity value of 20% evident after 0.6 ~ 0.8 g/kg calcium chloride addition. Previous studies reported similar calcium chloride-induced Chlase activation in plant leaves, which could be attributed to enzymatic conformational and surface charge changes, possibly promoting the association between enzymes and substrates. Li et al. (2011) found that 2 mmol/L and 7 mmol/L calcium chloride increased the Chlase activity in green tea leaves from 2.03 U/g to 2.71 U/g and 2.42 U/g, respectively. Gan et al. (2003) showed that calcium chloride addition increased the Chlase activity in wheat seedling leaves by 68%. The Chlase activity changes caused by calcium chloride may be related to the extraction and detection method, as well as the cultivars, pH, total solid level, and isoenzyme forms of Chlase. Furthermore, the Chlase activity decreased after adding calcium chloride concentrations higher than 0.7 g/kg. In comparison, 1.0 g/kg calcium chloride caused a nearly 10% decrease in Chlase activity. This was consistent with the work of Li (2011), who showed that 7 mmol/L of calcium chloride decreased the Chlase activity in green tea leaves by 52%. In conclusion, the effect of calcium chloride on the initial Chlase activity could be classified into three stages. Concentrations of 0 ~ 0.2 g/kg displayed no influence, 0.2 ~ 0.8 g/kg prompted activation, and 0.8 ~ 1.0 g/kg decreased activity. Therefore, 0.2 g/kg, 0.6 g/kg, and 1.0 g/kg calcium chloride concentrations were selected to examine the inactivation kinetics and thermal stability of the Chlase in mulberry leaves.
3.5 The effect of calcium chloride on the TP inactivation kinetics of mulberry Chlase
As shown in Fig. 3a and Figs. 6a, b, and c, the effect of calcium chloride on the thermal inactivation kinetics was remarkable. Although the experimental data of all four evaluated solutions (distilled water with 0 g/kg, 0.2 g/kg, 0.6 g/kg, and 1.0 g/kg added calcium chloride) could be described by the two-fraction kinetics (R2 = 0.950 ~ 0.995), their responses to thermal treatment were different. Calcium chloride enhanced the thermodynamic parameters and RA of the Chlase in the mulberry leaves (Table 3). The Chlase with 0.6 g/kg added calcium chloride displayed the highest thermal stability, followed by concentrations of 1.0 g/kg and 0.2 g/kg, as shown by the k and D values in Table 3, while they were all lower than the solution without calcium chloride (Table 1a).
Although adding 0.2 g/kg calcium chloride did not affect the initial Chlase activity, its inactivation during thermal treatment decreased compared to that without calcium addition. The inactivation constant rates of the sensible Chlase with 0.2 g/kg added calcium chloride ranged from 0.422 min− 1 to 1.344 min− 1 in a temperature range of 75 ℃~100 ℃, while 0.6 g/kg and 0.8 g/kg calcium chloride yielded values of 0.244 ~ 1.205 min− 1 and 0.409 ~ 1.288 min− 1, respectively. The corresponding DS values were 5.46 ~ 1.13, 9.44 ~ 1.36, and 6.78 ~ 1.19 min in the presence of 0.2 g/kg, 0.6 g/kg, and 1.0 g/kg of calcium chloride. The kR and DR trends for the resistant Chlase affected by calcium chloride were consistent with the sensible values.
The effect of calcium on enzyme thermal stability was reflected by a less pronounced RA decrease and higher thermodynamic parameters, including the Z and Ea values, in the solutions containing calcium compared with the distilled water without calcium (Fig. 6 and Fig. 7). The Chlase inactivation rate was over 99% after 6 min of thermal treatment at 100 ℃ in the absence of calcium (Fig. 3a), and approximately 90% in the presence of calcium. Although no studies are available that investigate how calcium chloride affects thermal Chlase inactivation-related changes, similar behavior was reported for the peroxidase (POD) in broccoli after adding 0.005 M of calcium chloride. Han et al., (2023) and Plieth and Vollbehr (2012) verified PODs activation and heat stabilization at very distinct calcium concentrations of 0.001 µmol/L to 100 mmol/L. Additionally, the Z values of the sensible and resistant Chlase with calcium were lower than those without calcium, which enhanced Chlase heat resistance. The verification experiments confirmed that adding 0.2 g/kg, 0.6 g/kg, and 0.8 g/kg calcium chloride exhibited similar goodness-of-fit results, while a good fit was evident between the predicted and experimental data in terms of the Af, Bf, SS, and RSME values (Table 2).
The increased activation and thermal stability of calcium-induced enzymes was possibly due to the protection on the active center since calcium ions could form salt bridges or prompt electrostatic interaction with amino acids or pectin to generate macromolecular complexes with higher stability (Veitch, 2004). Therefore, adding calcium chloride could reinforce the presence of the ions necessary to preserve the structural stability of the active enzyme center. Furthermore, Plieth and Vollbehr (2012) suggested the presence of two binding sites between calcium and enzymes. One was related to a catalytic domain, which promoted enzyme activity, while the other was associated with a stabilizing domain, increasing the thermal stability of the protein. In the present work, although 0.2 g/kg calcium chloride addition did not increase the Chlase activity, the heat resistance was higher, suggesting that 0.2 g/kg calcium did not reach the catalytic domain, mainly acting on the stabilizing domain. In addition to the binding sites, the increase in thermal stability might also be related to higher molecular surface tension during calcium binding (Plieth and Vollbehr, 2012). Moreover, enzymatic heat tolerance was previously assumed to be associated with the impact of aggregation since the bridges between the protein units increased the structural rigidity (Adalberto et al., 2010). Furthermore, calcium was recognized as ideal for structural protein stabilization, contributing to its ability to occupy irregular coordination environments with a wide variety of bond lengths and bond angles (Chigri et al., 2005). Therefore, molecular aggregation may be responsible for the calcium-related activation and increase in thermal stability of Chlase.