Long-term preservation of α-amylase activity in highly concentrated aqueous solutions of imidazolium ionic liquid

2.1 Materials

1-Butyl-3-methylimidazolium bromide, [C₄C₁im]Br, of high purity (>99%) was purchased from IoLiTec (Heilbronn, Germany).

The α-amylase enzyme (EC 3.2.1.1 from Bacillus licheniformis, Type XII–A) was supplied as a saline solution by Sigma (St. Louis, MO, USA) and was certified to contain 21.7 mg [protein] per cm³ (Biuret). Its current specific activity was measured by iodine-starch protocol [18], [19], [20], [21] and was found to be 3043 U per mg protein. One unit (U) is defined as the catalytic activity that hydrolyzes 1 μmol of starch to dextrins in 1 min at pH=6.00 (0.100 mol dm⁻³ Ac-buffer), at 30°C.

The salts, CH₃COONa×3H₂O (NaAc) and Na₂HPO₄×12H₂O, and the acid CH₃COOH were puriss. p.a., bought from the local market (Teocom, Sofia, Bulgaria). CH₃COOK (KAc) was a product of Sigma-Aldrich (St. Louis, MO, USA), K₂HPO₄ and KBr were products of Fluka (Buchs, Switzerland).

The substances required for the α-amylase activity assay (I₂, KI, and starch) were of analytical grade purchased from Teocom (Sofia, Bulgaria).

2.2 Preparation of the [C₄C₁im]Br/salt aqueous solutions containing α-amylase

In all solutions, the IL concentration was fixed at 3.660 mol dm⁻³, which is ca. 80.2% w/v (61.7% v/v). The salt added varied by type and concentration, as it is described below. The [C₄C₁im]Br/salt aqueous solution was stirred for an hour at room temperature. After that, the same amount of the α-amylase enzyme was introduced in each IL/salt aqueous solution, so that the final protein concentration was 0.27125 g dm⁻³. The solutions were stirred for 2 h prior to start of the analysis of the α-amylase activity, except for the shorter mixing of the solutions where a rapid deactivation was detected.

The pH values of all solutions were measured by a pH meter equipped with a pH combination electrode (Hanna Edge, Cluj-Napoca, Romania).

2.3 α-Amylase activity assay

The α-amylase activity assay was executed by the following steps. Aliquots of 0.050 cm³ of the [C₄C₁im]Br/salt aqueous solutions containing the α-amylase enzyme were withdrawn during the course of incubation (noted on the kinetics’ curves) and diluted 50-fold in 0.100 mol dm⁻³ NaAc. The diluted samples were stirred for at least 1 h. The pH values of all samples were 7.26±0.03, except for the diluted phosphate-systems, which were a little higher, 7.38 (Na₂HPO₄) and 7.76 (K₂HPO₄). That way the α-amylase was set at the optimal pH condition. The diluted samples were subjected to activity assay according to the procedure described by Pantschev et al. [19] and elsewhere [18], [20], [21].

The α-amylase activity measured during the course of incubation was evaluated as % from the initial activity in the same solution but without the IL and was assigned relative (residual) activity.

2.4 UV/VIS and fluorescence spectrophotometric measurements

The α-amylase enzyme incubated in the IL/salt aqueous solutions was characterized by UV-absorbance and fluorescence emission. The UV-spectra were recorded on an UV/VIS spectrophotometer (UV-1600PC, VWR, Leuven, Belgium) and the fluorescence measurements were carried out on a spectrofluorimeter Shimadzu (Model DR–15, Japan). The samples for both analyses were prepared by 4 times dilution (in 0.100 mol dm⁻³ NaAc) of aliquots from the IL/salt solutions containing α-amylase. In order to overcome the interference from the IL, all the samples were measured vs. their analogously prepared blank (without the enzyme) samples.

3.1 Kinetics of activity of α-amylase incubated in aqueous solutions of [C₄C₁im]Br/NaAc

Aqueous solutions containing [C₄C₁im]Br and NaAc were prepared. The IL-concentration was fixed at 3.660 mol dm⁻³, which is ca. 80.2% (w/v), while the NaAc-concentration was varied from 0 to 0.828 mol dm⁻³, the later being the limit of NaAc solubility in the solution. After that, the same amount of the α-amylase enzyme was introduced in each IL/NaAc aqueous solution. The pH values of the solutions were as follows: 5.31 (0 mol dm⁻³ NaAc), 7.14 (0.050 mol dm⁻³ NaAc), 7.66 (0.200 mol dm⁻³ NaAc), 7.89 (0.675 mol dm⁻³ NaAc), and 8.29 (0.828 mol dm⁻³ NaAc). The solutions were stirred for 2 h at 360 rpm and afterwards they were stored at room temperature. Aliquots were withdrawn therefrom during the course of time and subjected to α-amylase activity assay.

Results about the relative activity are presented in Figure 1A and B. It is clearly seen that a trace amount of NaAc (0.050 mol dm⁻³) in the concentrated [C₄C₁im]Br aqueous solution has a huge effect on the preservation of fully active α-amylase for 2-day grace period (Figure 1A). In the absence of NaAc, the enzyme was totally deactivated for the same period. The increase in the NaAc concentration to 0.200 mol dm⁻³ enlarges twice the grace period of full activity, for more than 4 days. At high NaAc concentrations, 0.675 and 0.828 mol dm⁻³, however, an opposite effect is observed (Figure 1B). Table 1 represents data about the periods of relative activity above 90% and above the half activity interpolated from the kinetics’ dependences. The shortening of the time of activity preservation in the more concentrated NaAc solutions could be explained in terms of reduced water activity (the water available to the enzyme).

Figure 1:

(A and B) Kinetics of activity of α-amylase during incubation in [C₄C₁im]Br aqueous solutions at different concentrations of NaAc.

Water is a key component of the nonconventional (predominantly nonaqueous) biocatalytic media [22]. Most biocatalysts need a certain amount of water even though the quantity required could seem surprisingly small [23]. This water is directly bound to the enzyme molecule. The water present in a solution could hydrate the other compounds but without contributing to the biocatalyst’s activity. Obviously, with increasing NaAc quantity, more water molecules become involved in the salt solubilization and less free water remains to hydrate the enzyme. As a result, the activity decreases. This hypothesis is afterwards worked out.

3.2 Kinetics of activity of α-amylase incubated in aqueous solutions of [C₄C₁im]Br and other salts

In addition to NaAc (0.828 mol dm⁻³), aqueous solutions of [C₄C₁im]Br and the salts Na₂HPO₄ and K₂HPO₄ were prepared in a way to obtain saturation with respect to the salt. The IL concentration was fixed at 3.660 mol dm⁻³, while the saturation with Na₂HPO₄×12 H₂O was reached at 0.115 mol dm⁻³ and with K₂HPO₄ – at 0.200 mol dm⁻³, the later borders the biphasic region of the aqueous system [C₄C₁im]Br/K₂HPO₄ [24], [25]. Then the α-amylase was added. The pH values of these aqueous solutions were 8.12 and 9.75, respectively. The three salts, NaAc, Na₂HPO₄, and K₂HPO₄, are commonly used to buffer the α-amylase solutions to desired pH values and normally could not negatively affect the catalytic activity. It is seen in Figure 2 that under limited hydration, both the IL and the salt strip the essential water from the biomolecule and its catalytic function is disturbed. In terms of inactivation, the system of the strongest kosmotrope (K₂HPO₄) is comparable to the solution without salt addition regardless of the mechanism of inactivation which is different, dehydration by the salt or formation of complex with the IL.

Figure 2:

Kinetics of activity of α-amylase during incubation in salt-saturated [C₄C₁im]Br aqueous solutions.

First-order kinetics model is widely used in the literature to investigate the enzyme deactivation [26], including by ILs [27]. The fast α-amylase inactivation in the highly salted IL solutions (Figure 2) was found to approach the first-order kinetics behavior (model resolution is presented as Supplemental Figure S1). Under the most severe (anhydrous) conditions in the system of K₂HPO₄, the kinetics of the α-amylase deactivation perfectly obeys the model. The case of “no salt” system, however, is not so simple because the inactivation curve seems biphasic, i.e. a rapid inactivation is followed by a decelerated decay. Such biphasic behavior is a relatively common phenomenon in enzymatic inactivation kinetics [28] and is observed in cross-linked or immobilized enzymes. According to Lencki et al. [28] the slow decay is most likely due to irreversible aggregation and coagulation reactions. Regarding the present α-amylase system, such irreversible interactions could be attributed to the precipitation that was observed to occur in the samples of the IL aqueous solution without salt addition (as discussed below).

It can be concluded that a microenvironment which provides the enzyme with an appropriate water hydration and acetate concentration, could serve as a shield for the α-amylase from being exposed to a rapid inactivation by the IL, and as a result the grace period is substantially enlarged.

Potassium acetate was also tested for comparison with sodium and the best long-term protection from inactivation (the longest grace period) was obtained. In addition, incubation in [C₄C₁im]Br/KBr aqueous solution, which contains anions of the same type, was carried out. All solutions contained same IL and salt (NaAc, KAc, or KBr) concentrations, 3.660 and 0.200 mol dm⁻³, respectively. The pH values of the Ac solutions were also equal, 7.66 (NaAc) and 7.68 (KAc). The pH of KBr system was 5.58. The results about the residual activity monitored during a month of incubation are shown in Figure 3. The periods of time for activity conservation above 90% and 50% are compared in Table 2. The change of sodium with potassium extends 3 times the time of full activity. The activity was retained above 90% for 2 weeks, and the half of activity remained in the end of a month. For the same period, the activity of the α-amylase dissolved in a regular (without IL) aqueous solution could be only stored under sterility as it fed easily the microbial growth. KBr, which matches the α-amylase enzyme in the ordinary aqueous solutions [20], [22], here did not protect the enzyme from being rapidly inactivated by the IL. A plausible explanation is discussed below.

Figure 3:

Kinetics of activity of α-amylase during incubation in [C₄C₁im]Br aqueous solutions with addition of different salts (0.200 mol dm⁻³), Na/KAc, or KBr.

3.3 Towards an understanding of the effect of the acetate salt on the α-amylase activity preservation in aqueous solutions of [C₄C₁im]Br

The UV-spectroscopy is an effective and simple tool to explore the structural change in protein molecules [29]. The UV-spectra of IL systems containing α-amylase of different degrees of activity are shown in Figure 4A and B and the respective profiles in the regular (without IL) solutions are also presented for comparison. The interactions of the IL with the enzyme reflect on the UV-spectrum which is altered in the near-visible wavelength range (Figure 4A) due to the suspended precipitates that can be seen in the sample. Precipitation was also observed in the sample of the inactivated solution of IL/KBr.

Figure 4:

(A and B) UV-spectra of α-amylase during incubation in [C₄C₁im]Br aqueous solutions.

A compact α-amylase conformation was revealed in the sample of IL/0.200 mol dm⁻³ KAc with 90% activity (2-week incubation) where no precipitation was observed (Figure 4B). The profile remained almost unchanged in a month of incubation except for the very slightly higher absorption in the near-visible range. The inactivation in the salty system of [C₄C₁im]Br/NaAc (0.828 mol dm⁻³) is obviously caused by complete unfolding of the enzyme. When protein unfolds a loosened conformation is developed where more chromophores become exposed to interactions with the solvent and as a result the absorbance increases [29], as observed in Figure 4B.

The alteration in the biomolecule’s conformation is thought to be responsible for the activity loss. Tertiary structural change of a protein chain induced by molecules could be detected by measuring the fluorescence spectra [29]. Most of the intrinsic fluorescence emissions from a folded protein are due to the excitation of Trp residues, which are sensitive to the microenvironment surrounding the fluorophores [30]. The fluorescence emitted by the α-amylase incubated in the aqueous solution of IL and in the IL/KAc was monitored and compared with the intensity in the absence of IL (Figure 5). A substantial reduction in the fluorescence emitted from the excited Trp fluorophores was observed, as expected, in the IL solution, where suspended precipitates were noticed and a rapid deactivation was measured. However, quenching was also observed in the sample of the active α-amylase. There was a negligible difference between the spectra of the fully active α-amylase (IL/KAc, 4th hour) and the α-amylase with half activity (IL/KAc, 30th day). This means that regarding the α-amylase enzyme, the Trp fluorescence quenching is not necessarily accompanied by an activity loss, as elsewhere stated [12].

Figure 5:

Fluorescence spectra of α-amylase during incubation in [C₄C₁im]Br aqueous solutions.

In future, in order to unravel the arrangement around the enzyme, diverse spectrometric studies must be carried out, which should consider the groups involved in the substrate binding sites on the α-amylase surface, such as histidine residues, as well as the groups of the active site [31], [32].

The results obtained show that the acetate anion acts as preventer for the α-amylase enzyme from being rapidly inactivated by the imidazolium cation of the IL. An important interaction must be considered to understand this effect. It is found that the Ac anion is able to accept a proton from C2–H on the imidazolium cation to generate a carbene, a highly energetic intermediate [33], [34]. This interaction might be involved in the mechanism of prevention from inactivation. In the presence of KBr, which is not capable of forming carbenes with [C₄C₁im]Br, a rapid inactivation was measured (see Figure 3) and precipitates were clearly seen in the samples, similar to the solution without salt. No precipitation was noticed in the samples of [C₄C₁im]Br/KAc during the long-term incubation. The acetate protects the α-amylase from being salted-out and precipitated by the IL.

Apart from physical methods applied to investigate the enzyme deactivation by the IL, an alternative methodology relies on the enzyme kinetic approach. By measuring the rate of the starch hydrolysis at different substrate concentrations in the presence of IL and acetate, the interaction between enzyme-IL/acetate or (enzyme-substrate)-IL/acetate could be deduced, as it has been recently shown in the case of cellulase and [C₄C₁im]Cl [27]. This constitutes a platform for a further work.