Structural evidence for kinetic and thermal stability changes of α-amylase due to exposure to [emim][lactate] ionic liquid

Materials

α-amylase, 1,3-dinitrosalicylic acid has been obtained from Sigma Chemicals Co. Potato starch and 1-ethyl-3-methylimidazolium L-(+)-lactate are from Sigma-Aldrich. All other chemicals are analytical grade. Solutions were prepared with double-distilled water.

α-Amylase assay

α-Amylases activity was determined according to the method developed by Miller (Miller 1959) with some modifications. In this method 3,5-dinitrosalisilic acid reagent was used for reducing sugars released by α-amylase. In brief, 0.5 mL of α-amylase enzyme (1 U/mL) with or without [emim][lactate] were added to 1 mL of starch solution (1% w/v in 20 mM sodium phosphate buffer with 6.7 mM sodium chloride, pH 6.9) and mixed by swirling and incubated for 15 min at 20 °C. Then, 1 mL of DNS solution (96 mM 3,5-dinitrosalicylic acid and 5.3 M sodium potassium tartrate) was added to the mixture and placed in a boiling water bath for 15 min to stop α-amylase activity. Solution were stored on ice for 10 min and then absorbance in 540 nm was recorded for sample. In the blank sample, α-amylase solution was replaced with the same volume of deionized water. One unit of α-amylase activity is defined as the amount of enzyme that releases 1 mM of reducing sugars (with maltose as the standard) per minute under the specified assay conditions.

Determination of kinetic parameters

The effect of [emim][lactate] concentration (1 and 0.4 M) on the Km and Vm parameters was evaluated by measuring the α-amylase activity in different amounts of starch (2–10 mg/mL) as substrate. The Lineweaver–Burk plot was used to determine Vm and Km factors.

Optimum temperature and pH

The optimum temperature of A. oryzae α-amylase in the absence and presence of [emim][lactate] was determined by the separate incubation of enzyme and starch solutions for 5 min at 25–80 °C (5 °C intervals). The solutions were then used for α-amylase assay.

α-amylase solution was prepared in different buffers with the desired pH (in range of 3–10) to investigate optimum pH. The following buffers were used: glycine-HCl, sodium citrate, sodium acetate, sodium phosphate, and tris-HCl. Then, the α-amylase solution with appropriate pH was used for enzymatic assay in the presence (0.4 M) or absence of [emim][lactate].

Thermal inactivation

Thermal inactivation of A. oryzae α-amylase was determined by the incubation of enzyme solution at 40, 50, 60, and 70 °C and was compared with same condition in the presence of 0.4 M of [emim][lactate]. Α-amylase activity of each sample was determined every 5 min for 20 min. Residual activity against time was used to compare the inactivation profiles.

Fluoresce spectroscopy

Intrinsic fluorescence of A. oryzae α-amylase (0.1 mg/mL) was examined in the absence and presence (0.2, 0.4, and 1 M) of [emim][lactate] using a Perkin-Elmer fluorescence spectrophotometer apparatus (USA). Enzyme samples were incubated for 5 min at room temperature, and then excited at 295 nm; emission was recorded from 300 to 400 nm. Both excitation and emission slits were set to 5 nm. A blank sample without enzyme was used to remove the background fluorescent effects of imidazolium ring of [emim][lactate]. The final emission spectra resulted from the subtraction of emission of each sample from the blank.

Effects of [emim][Lac] on the α-amylase activity

The effect of different concentrations (0.2–1 M with 0.2 intervals) of [emim][lactate] on α-amylase activity is represented in Figure 1. Relative activity of α-amylase shows decreases with increase in [emim][lactate] concentrations. Decrease in α-amylase activity in the presence of 0.2 M of [emim][lactate] was inappreciable. But α-amylase activity in the presence of 0.4 M of [emim][lactate] decreased to 60% compared to that in the absence of IL. The highest decrease (35% of initial reaction rate) was observed in the presence of 0.8 M of [emim][lactate]. Decrease in the activity of enzymes in aqueous solution containing IL and with increase in IL content is common [10], [25], [26], [27], [28]. Sate reports loss in the activity of Candida Antarctica lipase due to aggregation in the presence of [emim][EtOSO₃], [emim][NO₃] and [emim][N(CN)₂] [29]. Turner also reports [bmim][cl]-induced inactivation and unfolding of cellulase from Trichoderma reesei [30]. However, in our previous study, the activity of luciferase from Photinus pyralis was observed to increase in the presence of [TMG][Lac] [31]. Moreover, H-bonding basicity and nucleophilicity of anionic part of ILs affect the enzyme activity in the presence of ILs [32]. In addition to the anionic part, other factors—including alkyl chain length in cations, IL polarity, viscosity, ion kosmotropicity, amphiphilicity, hydrophobicity, and an IL network—can affect enzyme, medium, and substrate relationship and hence enzyme activity [33]. Comparison of the activity of commercial peroxidase in the presence of five 1-alkyl-3-methylimidazolium-based ILs represent better activity in the presence of ILs with short alkyl chain length [34]. Therefore, there is no general rule to evaluate and predict the changes in the activity of enzymes in the presence of different ILs; hence, these effects should be examined.

Figure 1:

The effect of different concentrations of [emim][Lac] on α-amylase activity.

Effects of ionic liquid on the kinetic of enzyme

Michaelis–Menten and Lineweaver–Burk plots were used for the determination of Vm and Km of α-amylase from A. oryzae in the absence and presence of two concentrations (0.4 and 1 M) of [emim][lactate]. The hyperbolic curves are the characteristic feature of a true enzyme. The obtained values for Vm and Km of α-amylase are listed in Table. Km increases from 0.005 M in the absence of [emim][lactate] to 0.019 and 0.042 in the presence of 0.4 and 1 M of [emim][lactate] respectively. This increase reveals the decrease in affinity and looser substrate binding by enzyme [35]. Unlike Km, Vm decreases in the presence of both concentrations compared to the absence of [emim][lactate] (see Table 1).

Optimum temperature and pH of a-amylase

The sensitivity of α-amylase to temperature (25–80 °C) in the absence and presence of [emim][lactate] (0.4 M) was compared; the results are shown in Figure 2. α-amylase activity in the absence of [emim][lactate] reached its maximum at 45 °C. In light of the fact that 98% of the activity is at 50 °C, the optimum temperature can be determined at 45–50 °C. In the presence of [emim][lactate], maximum activity is achieved at 50 °C. According to these results, there is no significant change in the optimum temperature of α-amylase in the absence and presence of α-amylase.

Figure 2:

The activity of α-amylase with and without [emim][Lac] at different temperatures from 25 to 80 °C.

The pH profile of α-amylase in the presence and absence of [emim][lactate] is depicted in Figure 3. According to this, the optimum pH of α-amylase decreased from 8.5 in the absence to 7 in the presence of [emim][lactate]. Electrostatic interactions between ionized side chains of amino acid residues play a critical role in the overall structure of proteins and therefore their activity. The ionization state of these residues depends on the pH of their environment. Therefore, the changes in environmental pH can change the activity of enzymes. These effects of hydrogen ion concentration on the activity of enzymes have close similarities to the effects of activators and inhibitors. The pH profile of enzyme activity can yield valuable information on the nature of the kinetic mechanism followed by the enzyme and can help to identify specific groups playing important roles in the reaction [36]. Different factors affecting the pKa of side chain of residues in proteins, including dehydration (Born effect), charged-charged interactions (Columbic interactions), and charged dipole interactions (hydrogen bonds), are most important [37]. Solvents can change the pKa values of ionizable groups in proteins by interfering with the natural environment of amino acid residues. Therefore, shifts in optimal pH value of enzymes due to the change in the solvent system can be seen.

Figure 3:

Relative activity of α-amylase against pH in the absence and presence of [emim][lac].

Thermal stability of a-amylase

One of the major goals of the use of ILs as reaction media for enzymes is the improvement of the thermal stability of enzymes. This approach has advantages such as relatively low cost- and time-saving compared to other methods such as immobilization and genetic engineering. Using ILs to improve the thermal stability of enzyme is not always successful. Apart from the reports in the stabilization of enzymes, there are some observations of decrease in enzymes’ thermal stability. Therefore, enzymes are not homogeneously stable in all types of ILs; the choice of correct IL is the most important factor [38]. In this study, thermal inactivation of A. oryzae α-amylase at 40, 50, and 60 °C is determined by incubating enzyme solutions in the absence and presence of 0.4 M of [emim][lactate]. The α-amylase activity assay was performed every 5 min for 20 min using an incubated enzyme solution. According to the results (Figure 4), activity of α-amylase shows decrements in all temperatures in the absence and presence of [emim][lactate]. Inactivation rates of α-amylase in both conditions are quite similar at 40 °C and there is no significant difference at 50 °C. The rate of inactivation at 60 °C is obviously different in the absence and presence of [emim][lactate]. In this temperature, α-amylase in the presence of [emim][lactate] loses its initial activity up to 80% after 20 min, while in the absence of [emim][lactate], it just loses 40% of its initial activity in the same time. Similarity in the inactivation rates at 40 and 50 °C may be related to the range of optimum temperature of the enzyme in the absence and presence of [emim][lactate]. Similar results are reported by other studies. Dabirmanesh reports a decrease in the activity and thermal stability of two related a-amylases from Bacillus amyloliquefaciens and Bacillus lichiniformis in the presence of [BMIm][Cl] and [HMIm][Cl] [10]. Heller reports that thermal stability of green fluorescent protein (GFP) decreases in the presence of [bmim][Cl] [39].

Figure 4:

Percentage of relative remaining activity vs time plots to investigate thermal inactivation of α-amylase in the absence (•) and presence (▴) of 0.4 M of [emim][Lac] at 40 (a), 50 (b) and 60 °C (c).

These results, alongside our results, show that ILs do not only increase the thermal stability of proteins but can also decrease their thermal stability.

Intrinsic fluorescence spectroscopy

Fluorescence spectroscopy is usually used to study folding/unfolding, substrate binding, and external quencher accessibility in proteins. One of the major advantages of using intrinsic fluorophores is that proteins are in their native state and there are no structural changes due to the addition of external fluorophores. Intrinsic fluorescence based on the Trp residue(s) is a simple and reliable method that is usually used for studying the folding/unfolding states in proteins. The λ_max of Trp residue is quite sensitive to its local environment and the degree of exposure to solvent [40]. This technique is used in this study to investigate the structural transitions of α-amylase due to its exposure to the different concentrations of [emim][lactate]. Four concentrations (0.2, 0.4, 0.6, and 1 M) of [emim][lactate] were used to acquire more information about the structural changes. The results of this experiment have been shown in Figure 5. As can be seen, α-amylase exhibited the same λ_max in the absence and presence of [emim][lactate] but maximal fluorescence intensity (I_max) was drastically decreased when exposed to the [emim][lactate]. Decrement in the I_max continued with increase in the concentration of [emim][lactate] and reached nearly zero in the presence of 1 M of [emim][lactate]. Decrease in the I_max of Trp residue represents the unfolding of the structure of protein and exposure of Trp residues to the more polar environment. This evidence reveals the reason of the decrease in the activity of the α-amylase accompanied by the increase in the concentration of [emim][lactate] (Figure 1). The EMIm cations interact with lactate anions and generate a network of interactions that is similar to that of the three-dimensional network of interactions between water molecules. Lactate anion has great hydrogen bond capacity and hence a greater basicity and stronger nucleophilicity. These properties interfere with internal hydrogen bonds and interactions with the positively charged sites in α-amylase; therefore, the α-amylase conformation changes to the unfolded state.

Figure 5:

Intrinsic fluorescence analysis of α-amylase in the absence and presence of different concentrations of [emim][Lac].