1. Introduction
The study of L-asparaginases (L-ASNases; E.C. 3.5.1.1.) from thermophilic sources is an important task that aims to improve the properties of enzymes for application in the food and pharmaceutical industries. L-ASNase is commonly used as a processing agent in the food industry to reduce acrylamide levels in commercial fried foods while maintaining their color, flavor, and texture [
1,
2]. L-ASNase catalyzes the hydrolysis of L-asparagine (L-Asn), which helps to prevent its reaction with sugars and the formation of acrylamide. Acrylamide is a potentially carcinogenic substance formed when reducing sugars react with L-Asn (Maillard reaction,
Figure 1) at temperatures above 100–120 °C during frying, baking, and roasting [
3,
4,
5]. To reduce the concentration of L-Asn in products, foods containing high amounts of sugars can be treated with L-ASNase. This enzymatic treatment can result in less acrylamide formation during food preparation.
However, the stability of existing commercial L-ASNase is limited, which may affect their application in certain fields.
Aspergillus fungi are commonly used in the baking industry, but their enzyme activity may decrease at temperatures above 100–120 °C [
7]. Hence, one of the main issues is the search for new sources of L-ASNases with improved thermal stability. It is worth considering hyperthermophilic organisms, such as the archaea
Thermococcus sibiricus, as potential sources for this purpose [
8]. This L-ASNase from archaea has optimum activity at 90 °C and can be used for industrial applications.
One approach to regulating the catalytic properties and thermostability of enzymes is through the formation of conjugates with polymers [
9,
10]. Immobilization prevents denaturation and aggregation of the protein at elevated temperatures and preserves its activity. This was successfully achieved upon covalent modification of L-ASNases from different sources [
11,
12,
13,
14,
15]. Previous studies have demonstrated the efficacy of modifying the L-ASNases of
Erwinia carotovora (EwA) and
Rhodospirillum rubrum (RrA) with polycations such as graft polymers based on chitosan and polyethylenimine (chitosan-PEG, chitosan-glycol, PEI-PEG, chitosan-PEI) [
16,
17,
18]. Polyamines were observed to be effective at modifying RrA. The conjugation of RrA with PEI derivatives, including PEI-PEG, resulted in higher activity at pH 7.5, improved thermostability, and increased resistance to trypsinolysis in comparison to the unmodified enzyme. The aim of this study was to investigate the possibility of regulating the catalytic properties of
Thermococcus sibiricus L-ASNase by producing conjugates of the enzyme with polymers of different compositions. This study compared the effects of PEI, PEG, and spermine on the structural properties, activity, and thermostability of the enzyme.
3. Discussion
The modification of L-ASNases with different polymers could enhance their physicochemical and biocatalytic properties for industrial and medical applications. Our studies aimed to improve the catalytic parameters of the enzyme via modification and also to increase its stability to aggregation.
To date, the most common approach for the regulation of the catalytic parameters of the enzyme and the increase in its stability is PEGylation. To obtain a stable bond between proteins and PEG, activated PEG derivatives containing heterocyclic compounds, such as cyanurchloride and succinimide, are used, allowing a reaction between PEG and protein at mild pH and temperature values. PEGylation is a well-developed technology and is used in biopharmaceuticals to increase stability and solubility and improve the immunological properties of biologically active compounds. Among pegylated enzyme-based preparations, hydrolase-class enzymes are most widely used. Relevant examples include PEG-arginase, PEG-uricase, PEG-carboxyoxidase A, PEG-staphylokinase, PEG-glutaminase, PEG-asparaginase, etc. [
23,
24]. Recently, asparaginase from E. chrysanthemy has also been pegylated. Now, this drug is in the stage of preclinical studies. PEG is known to assist in stabilizing the enzyme’s secondary structure and preserving its natural conformation. The polymer is used for the modification of L-ASNase, and PEGylated L-ASNase preparations are already commercially available [
25,
26,
27].
Additionally, polycations are commonly used to modify enzymes, the effects of which vary depending on the polymer’s structure and the enzyme itself [
28,
29]. Polyelectrolytes can be used to change the charge of an enzyme, which can affect its properties. For instance, by modifying the charge near the active center, enzyme activity can be altered. This approach can be advantageous for achieving higher activity within the desired pH range. For example, polyamines have been used to shift the pH optimum of an enzyme toward more neutral values to increase the cytotoxicity of RrA [
17]. It has been suggested in the literature that the cytotoxicity of enzyme preparations can be influenced by increasing L-Asn hydrolysis activity at pH values at which tumor cells grow. Furthermore, the adsorption of the enzyme on tumor cell membranes can be further affected by polycations, which can have an impact on cytotoxicity.
Recently, polyamines, such as chitosan and PEI derivatives, have been found to be effective at modifying RrA, possibly contributing to the improvement in the abovementioned properties [
17]. However, since enzymes from different sources have different structures and properties, the effects of polymers may also differ, as in the case of EwA and RrA [
18]. This study aimed to compare the effects of charged (Spm and PEI) and uncharged (PEG) polymers on hyperthermophilic TsA with the effects on other L-ASNases that we have recently studied.
Upon studying the influence of the conjugate formation with polymers, an important parameter affecting the catalytic properties of the enzyme is the oligomeric composition. It is believed that L-asparaginases from hyperthermophilic sources can exist as monomeric or dimeric forms [
30,
31,
32]. Here, the chromatograms for the TsA enzyme studied reveal only a monomeric form; the dimer is not formed under the conditions studied. SDS-PAGE analysis of the TsA structure revealed a major band at approximately 37 kDa, which corresponds to the monomeric form (
Figure S1). Additionally, a very minor band at around 80–90 kDa was observed, which is likely to be the dimeric form. This form is more manifested when the enzyme is incubated with oxidized glutathione (
Figure S1A). Native electrophoresis (
Figure S1C) also shows a major band corresponding to the monomeric form of the enzyme and a minor band with a higher molecular weight, corresponding to the dimer. The monomeric form still predominates, with a content of at least 90%. To initiate dimeric form formation and the aggregation process, the enzyme was incubated at 90 °C up to 6 h, and as a result, the bands corresponding to the dimeric form and aggregates were detected using SDS-PAGE (
Figure S1B). However, it is worth noting that the dimeric form is not observed in the HPLC chromatogram (
Figure 2). To further examine the oligomeric composition, TIC and UV chromatograms (
Figure S2) as well as mass spectra of TsA solutions (
Figure S3) were obtained. Based on the TIC and UV chromatograms and the analysis of the mass spectra (
Figure S3), it appears that only the monomeric form is present in solution.
Analysis of TsA conjugates by HPLC chromatography as well as SDS-PAGE analysis were carried out to control the oligomeric composition of the TsA upon conjugate formation. For TsA-PEG in the HPLC chromatogram (
Figure 2), the main major peak was assigned to the PEGylated enzyme. SDS-PAGE analysis for TsA-PEG also showed one major blurred band around 73–94 kDa (
Figure S4). This band is most likely attributed to the monomer with PEG chains, which consists of approximately 5–7 PEG chains (25–35 kDa) and TsA monomers (37.5 kDa), resulting in a total of 62–72 kDa.
It is also important to mention that conjugates of L-asparaginase with polycations typically do not have significant differences in secondary structure from the native enzyme [
17]. In the case of TsA, there was no significant change in the CD spectra after conjugate formation. Therefore, it can be concluded that neither PEGylation nor modification with polyamines affects the secondary structure of TsA.
When analyzing the thermal inactivation for native TsA and conjugates with PEI and Spm, we observed initial regions within 20 min corresponding to first-order inactivation. After 20 min of thermoinactivation, the increase in the order of the inactivation constant is observed. This suggests a multimolecular process—aggregation—which was also observed by other methods such as CD, fluorescence, and visual observation. So, the enzymes inactivated via denaturation, followed by aggregation processes. All samples studied, except TsA-PEG, exhibit aggregation after a 20 min interval.
The fluorescence data appear to correlate with the thermoinactivation data (in terms of catalytic activity) for the samples studied. The thermograms show that, in the initial phase, the loss of the tertiary structure of the enzymes is predominant, followed by the destruction of the secondary structure (at temperatures above 80–85 °C according to CD spectroscopy) and the formation of aggregates, some of which are insoluble (
Figure 9C). In the case of TsA-PEG, no precipitation was observed. However, a loss of activity was detected, which seems to be mainly linked to the disruption of the tertiary structure.
As a result, the impact of polyelectrolytes on various L-ASNases with different structures and properties may vary. For example, when studying the thermoinactivation of the polyelectrolyte complex of EwA with PEI, it was found that the thermoinactivation constant for this PEC is higher than that for the native enzyme [
18]. The destabilization of the EwA structure can be attributed to the excessive positive charge of PEI. On the other hand, RrA complexes with PEI exhibited higher thermostability, as indicated by a decrease in the thermal inactivation constant. It has been previously observed that polycations may also contribute to the stabilization of the quaternary structure of RrA [
17]. A decrease in the aggregation degree and
kin of the TsA-PEI conjugate was also observed in comparison with those of TsA. Therefore, conjugation with Spm and PEI significantly stabilizes the enzyme against thermoinactivation. The thermoinactivation of the TsA-PEG conjugate, which alters the inactivation mechanism, is an interesting result. For this conjugate, no protein aggregation was observed after heating at 88 °C. However, in the case of TsA-PEG
kin, there was a decrease in activity, although the conjugate retained its secondary structure but can lose its tertiary structure. This phenomenon has also been observed for RrA [
21]. In the case of TsA, PEG may help protein particles repel each other better at elevated temperatures due to the presence of long hydrophilic PEG chains.
Therefore, our study demonstrated the prospects of the approach for regulating the biocatalytic properties and stability of enzymes of the biotechnological application on the example of the L-asparaginase TsA. The developed approach is based on the formation of conjugates of enzymes with PEG and grafted copolymers of a branched structure based on polycations. The polyelectrolyte nature of the polymers promotes their multipoint electrostatic interaction with the protein surface, which makes it possible to modulate the catalytic properties of the enzyme, including by shifting the pH of the optimum enzyme activity to the range of physiological pH values (pH 7.5–8.5). Optimization of the molecular architecture and composition of conjugates led to an increase in the catalytic efficiency of the enzyme (Vmax/KM) up to 1.5 times (
Table 4). The most striking effect on the catalytic activity of the enzyme is the modification with spermine. One of the main reasons for the change in conjugate activity in comparison with the native enzyme is the shift in the optimum pH of enzyme activity toward physiological pH values. In addition, the formation of conjugates with PEG leads to an increase in thermal stability and stability to thermoinactivation, compared with the native enzyme, reducing its inactivation constant by ~1.5 times, depending on the composition of the conjugate.
4. Materials and Methods
4.1. Enzyme Preparation and Chemicals
The enzyme preparation of LASNase from the microorganism
Thermococcus sibiricus was performed according to a method described previously [
8]. The L-ASNase gene of
Thermococcus sibiricus (sequence 1510265–1511260
https://www.ncbi.nlm.nih.gov/nuccore/NC_012883.1, accessed on 4 April 2024, protein GenBank accession No. WP_015849943.1) was synthesized by TWIST Bioscience (Twist Bioscience HQ, San Francisco, CA, USA). The synthesized gene was cloned and inserted into the pET-28a(+) vector under the control of the T7 promoter. The constructed vector was subsequently transformed and expressed in
E. coli BL21 (DE3) cells.
Selected recombinant
E. coli clones were grown as previously described [
17]. To culture the cells harboring the plasmids, 0.05 mg/mL of kanamycin was added to the medium. Expression of the target protein was induced by lactose added to the expressed culture at an OD600 of 1.9 to a final concentration of 0.2%. The cells were grown for another 17–20 h and then centrifuged at 4000×
g for 15 min.
All the enzyme purification steps were carried out at +4 °C. Five grams of biomass was suspended in 50 mL of buffer (20 mM sodium phosphate buffer (pH 7.2), 1 mM glycine, and 1 mM EDTA) and ultrasonicated. Cell debris and nondegraded cells were removed by centrifugation (35,000× g, 30 min). The supernatant containing the target enzyme was applied to SP-Sepharose columns. The proteins were eluted with a linear gradient of 0–1.0 M NaCl. The protein content of the fractions was checked by absorbance at 280 nm and by measuring enzyme activity. Ultrafiltration, desalting, and buffer exchange were performed using Amicon membranes (Millipore, Burlington, MA, USA). The samples were frozen and stored at −20 °C.
The following reagents were used in this work: L-asparagine (BioChemica, Billingham, UK), phosphate-salt buffer and sodium tetraborate (Na2B4O7-10H2O, (PBS; Eco-Service, Moscow, Russia), citric acid and NaOH (Reachim, Moscow, Russia), DMSO (Sigma-Aldrich, Burlington, NJ, USA).
4.2. Synthesis and Purification of L-Asparaginase Conjugates
The following reagents were used for synthesis: linear polyethyleneimine, 2 kDa (PEI; Sigma-Aldrich, St. Louis, MI, USA); spermine (Spm; Sigma-Aldrich, USA); and linear activated Methoxy PEG Succinimidyl Carboxymethyl Ester 5 kDa (PEG; JenKem, Zhengzhou, China).
L-ASNase reaction with activated PEG: To a mixture of 5 mg/mL of TsA in 10 mM PBS (pH 7.3; ECO-Service, Russia), a 15–25-fold excess of PEG was added, and the mixture was dissolved in a minimal volume of DMSO. The resulting concentration of DMSO in solution with protein did not exceed 5–10%. The resulting mixture was incubated under stirring at room temperature for 2 h. The product was purified by diafiltration on Amicon centrifuge filters® (Merck-Millipore, Kenilworth, NJ, USA) with pores allowing molecules less than 30 kDa in mass to pass through. Filtration was carried out approximately 7 times. The determination of the number of PEG chains bound to the protein was carried out using IR spectroscopy. Furthermore, similar results were obtained through the application of the titration method with TNBS. Additionally, an AFM microscope (NTEGRA II, NT-MDT Spectrum Instruments, Moscow, Russia) was used to control the modification and visualize polymeric conjugates of the enzyme and compare it with non-modified form.
L-ASNase was reacted with Woodward K reagent (WRK, Sigma-Aldrich, Burlington, NJ, USA) and polyamines. To a solution of TsA in 50 mM MES (pH 5.5), a solution of WRK reagent in HCl (Component-Reaktiv, Moscow, Russia) (pH 2.0) was added to achieve final concentrations of 1 mg/mL (0.027 mM) and 0.5 mM, respectively. The pH 5.5 of the reaction solution was controlled by MES buffer (pH 5.5). The resulting mixture was incubated for 2 h at room temperature, after which the excess WRK was removed through diafiltration. The completeness of the purification was monitored by observing the decrease in peak intensity at 245 nm. A 30-fold excess of PEI relative to the mol of enzyme was added to the purified TsA-WRK adduct in 10 mM PBS, pH 7.3. The final concentrations of ASNase and PEI in solution were approximately 1–1.5 and 1.5 mg/mL, respectively. The mixture was incubated for approximately 15–20 h at room temperature. The conjugate was subsequently purified by diafiltration approximately 4–5 times. The purity of the preparation was assessed using HPLC gel filtration on a Superdex 200 Increase 10/300 GL column in a Knauer chromatography system (Knauer, Berlin, Germany). The eluent consisted of 50 mM Tris buffer and 200 mM NaCl at pH 7.5, with an elution rate of 0.75 mL/min at 25 °C. Bovine serum albumin (BSA, Sigma-Aldrich, Burlington, NJ, USA) with a molecular mass of 66.4 kDa was used as a reference protein. The resulting conjugates were either lyophilized or frozen and stored at −20 °C.
4.3. L-Asparaginase Catalytic Activity Measurement
The enzymatic activity of the L-ASNases was measured on a Jasco J-815 circular dichroism (CD) spectrometer (Jasco, Tokyo, Japan) according to the method described previously [
17]. The reaction was carried out by mixing solutions of L-asparagine and L-ASNase in 25 mM borate buffer at pH 9.3 to final concentrations of 1–40 mM and 0.007–0.01 mg/mL (0.2–0.3 μM), respectively. The change in ellipticity was recorded at a wavelength of 210 nm in three replicates. The reaction was performed in a 300 μL quartz cuvette with an optical path length of 1 mm in a temperature-controlled cell at 85 °C. The dependence of the initial reaction rate on the substrate concentration was plotted using Prism 8 software (GraphPad Software, San Diego, CA, USA). The values of Vmax and K
M were determined by linearizing the experimental data in a Lineweaver–Burk plot (or double-reciprocal plot, 1/V, 1/S
0).
To determine the pH dependence of the native enzyme and its conjugates, a series of solutions containing 20 mM L-asparagine in 5 mM citrate–phosphate–borate buffer were prepared. The pH of the L-asparagine solutions was adjusted to a range of 5.5–11 using NaOH solution. Next, 10 μL of enzyme at a concentration of 1 mg/mL was added to 300 μL of substrate solution in the cuvette, and the activity was recorded at 37 °C using a CD spectrometer.
The temperature dependence of L-ASNase activity was determined using a Peltier cell line, PMH-428s/15, which heats CD spectrometer cells. In a typical experiment, 300 µL of a preheated substrate solution (20 mM L-asparagine, 25 mM borate buffer, pH 9.3) was used. Next, 10 µL of enzyme solution was added, and the activity was measured at temperatures ranging from 35 to 95 °C. Product accumulation curves were recorded for 100–200 s.
Thermal inactivation curves of L-ASNases were obtained using a previously described method [
18]. In a typical experiment, a sample enzyme solution (0.5 mg/mL) was incubated in 10 mM PBS (pH 7.3) at 88 °C. After every 2.5–10 min of incubation, aliquots were taken and cooled for 4–5 min to room temperature. The enzymatic activity of the samples was measured at 85 °C. The obtained data were linearized in first-order reaction coordinates.
4.4. Registration of CD Spectra
CD spectra of the L-asparaginase solutions and purified conjugate solutions were recorded using a J-815 CD spectrometer (Jasco, Tokyo, Japan) equipped with a thermostatically controlled cell. Measurements were performed in the wavelength range of 200–260 nm at 37 °C in a quartz cuvette (l = 1 mm). Spectra were obtained by 3-fold scanning in 1 nm steps. Then, 300 μL of enzyme and conjugate samples in 10 mM PBS (pH 7.5) were added to the cuvette. The final concentration of native enzyme in the system was 0.25–1.5 mg/mL. The concentration of protein in the conjugates was determined from the graduation curve for the native enzyme. The data were analyzed using Prism 8 software (GraphPad Software, San Diego, CA, USA). Deconvolution of the spectra to analyze the content of secondary structures was performed using CDNN 2.1 software (Applied Photophysics Ltd., Surrey, UK).
4.5. Registration of IR Spectra
A Tensor 27 IR Fourier spectrometer (Bruker, Ettlingen, Germany) with an MCT detector was used to obtain the FTIR spectra of TsA and its conjugates. The measurements were carried out with a BioATR II thermostated cell using a single-reflection ZnSe element at 22 °C and FTIR microscope MICRAN-3. An aliquot (40 µL) of the corresponding enzyme solution (1.0–1.5 mg/mL in 10 mM sodium phosphate buffer) was applied to the internal reflection element, and the spectrum was recorded three times in the range from 3000 to 950 cm
−1 with a resolution of 1 cm
−1; we performed 50-fold scanning and averaging. The background was registered in the same way and automatically subtracted by the program. The resulting spectra were smoothed by the Savitzky–Golay method to a spectral resolution of 2 cm
−1 [
33]. The spectra were analyzed using Opus 7.0 software (Bruker, Ettlingen, Germany).
4.6. Registration of Fluorescence Spectra
Fluorescence spectra in the 290–390 nm range were obtained on a Cary Eclipse Fluorescence spectrometer (Agilent Technologies, Santa Clara, CA, USA) with an excitation wavelength of 280 nm (for Trp and Tyr excitation). TsA and conjugate solutions were prepared in 10 mM PBS to a final concentration of 1 mg/mL. The obtained solutions were heated to the required temperatures in a thermostatically controlled cell for 1–2 min. Then, measurements were performed. To determine the T50 parameter, graphs of the dependence of fluorescence intensity at 320 nm on temperature were plotted and normalized using Prism 8 software.