water interface

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Journal of CO2 Utilization journal homepage: www.elsevier.com/locate/jcou

Molecular dynamics simulation of high-pressure CO2 pasteurization reveals the interfacial denaturation of proteins at CO2/water interface Hassan Monhemia, Samaneh Dolatabadib, a b

⁎

Departement of Chemistry, Faculty of Sciences, University of Neyshabur, Neyshabur, Iran Departement of Microbiology, Faculty of Sciences, Neyshabur Branch, Islamic Azad University, Neyshabur, Iran

A R T I C LE I N FO

A B S T R A C T

Keywords: Cold pasteurization High pressure carbon dioxide Interfacial denaturation Molecular dynamics simulation

While mirobial and enzyme inactivation by CO2 is a promising non-thermal method for cold pasteurization of liquid foods, the poor understanding of the underlying molecular mechanism limits the development of this procedure. The complexity of the process along with the high-pressure conditions have disabled the experimental techniques to establish microstructural information for this phenomenon. In this work, molecular dynamics simulation method was used to find microscopic insights about this system. Myoglobin protein and lysozyme enzyme have applied as models in the simulations. It was found that CO2 and water make a distinct biphasic system in the experimental pressures of pasteurization process. Although many hypothesizes have attributed the enzyme inactivation to the solubilized CO2 molecules in the aqueous phase, we have shown that enzymes can be inactivated by an “interfacial denaturation” mechanism. Results show that the protein migrates from pure aqueous phase to the CO2/water interface and becomes denatured there. The molecular mechanism includes releasing the hydrophobic cores to the CO2 phase and escaping of the hydrophilic surface residues to the aqueous phase. These two phenomena denature the protein to a flat and extended conformation. Moreover, chemotrypsin inhibitor 2 protein was used to obtain the effect of distance from interface in the denaturation process. It was found that the distance from interface is critical in both performance and rate of the denaturation. Our simulations not only shed some lights on the molecular mechanism of CO2 pasteurization methods, but it can also be a basic computational model for further technological design and development.

1. Introduction High-pressure carbon dioxide (HPCD) [1] as a green non-thermal sterilization method [2,3] is regarded as an efficient tool for inactivation of enzymes [4–6] and microorganisms [3,7–9] at low temperatures. In HPCD system, either high pressure sub-or supercritical CO2 (scCO2) were applied in a batch or continuous method [1]. ScCO2 is an especial phase of CO2 which exists above its critical point temperature and pressure (Tc=31.1 °C, Pc=7.38 MPa). In the other hand, when temperature and pressure are below its critical point, CO2 is in subcritical state. HPCD is commonly used in the processing of the different liquid foods such as fruit juices [4,10,11] and dairy products [4,6]. This method is also very useful in the sterilization of the heat-sensitive biomedical materials at mild conditions [12,13]. Different enzymes such as pectinesterase [3–5,9,14], glucoamylase [6,15], acid protease [6,8,15], lipase [6,15], amylase [8,15,16], lipoxigenase [7], horseradish peroxidase [17,18], polyphenoloxidase [2,18–21], and phosphatase [22] were inactivated in HPCD process. Despite the advantages

⁎

of this process, there are many doubts about inactivation mechanism of enzymes and microorganisms in HPCD, which limits further technological developments. The complications become increased by dual behaviors of compressed CO2 in enzymatic reactions. Interestingly, while CO2 in HPCD process helps to inactivate enzymes, in scCO2 it is an enzyme activator [23–26]. In both processes, CO2 is used at high pressures and can interact with enzymes microstructure. The role of CO2 molecules in these two opposite processes is ambiguous. The conclusions about the using CO2 in enzyme activation is also very controversial. Some reports showed the usefulness of enzymatic reactions in presence of CO2 molecule [27,28] while others argued that it may adversely effect on the enzymes [29,30]. There is no general agreement about the impact of CO2 on the enzyme structure and activity [25]. Most of indeterminacies in this filed are due to the blindness of the different experimental methods in the investigation of HPCD as a very heterogeneous process. The processing vessel of HPCD commonly includes a liquid with the different biochemical contents in equilibrium with CO2 phase, which makes a multiphasic biochemical/aqueous/

Corresponding authors. E-mail addresses: [email protected] (H. Monhemi), [email protected], [email protected] (S. Dolatabadi).

https://doi.org/10.1016/j.jcou.2019.10.004 Received 10 July 2019; Received in revised form 3 October 2019; Accepted 4 October 2019 2212-9820/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Hassan Monhemi and Samaneh Dolatabadi, Journal of CO₂ Utilization, https://doi.org/10.1016/j.jcou.2019.10.004

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

2.2. Algorithms

supercritical system. Moreover, the reaction vessel is pressurized to the high pressures up to about 30 MPa. Molecular level investigations by practical tools are very challenging in such complex and harsh conditions. Molecular Dynamics (MD) simulation is a computer simulation technique, which shows the atomistic movement of a system based on Newtonian mechanics, interatomic potentials, and force fields [31]. It is regarded as a computational microscope in molecular biology [32]. Capturing of the structural and dynamical behavior of proteins and many biological macromolecules in full atomic detail becomes possible using MD simulations. This method has been frequently used in protein folding researches [33] and drug discovery [34]. Applications of MD simulations in the field of food processing are rapidly increased during recent years [35]. Many successful examples were reviewed by Chen et al. [36]. During recent years, we have investigated the effects of scCO2 on the structure and function of the model enzymes and proteins using MD simulations [37–43]. These studies reveal many microstructural properties and unknown aspects of the proteins in dry scCO2, which were in reasonable agreements with the experimental observations. MD simulations not only proved to be applicable in revealing poorly understood stabilization mechanisms [44], but it also can be applied in exploring new methods for stabilizing proteins and enzymes in natural solvents [45]. However, most of these works have focused on the enzyme activation and stabilization while HPCD is commonly used for enzyme inactivation purposes. Moreover, these simulations mostly were about the monophasic scCO2 and not biphasic system of HPCD. To our knowledge, there is no simulation data about the effects of HPCD system on proteins and enzymes. In this work, HPCD including a biphasic system of water and carbon dioxide and its effect on protein structure and stability is simulated for the first time. Applying MD simulations could be very helpful at least in two aspects of HPCD process. Firstly, the unknown nature of the processing vessel, especially at the molecular levels, has confused many researchers to design appropriate conditions to improve their process. Moreover, they did not know about the fragility of the different enzymes in HPCD. MD simulations of the process can give an atomistic view, which responds to the unsolved questions about this process. Secondly, by establishing a fundamental dynamics model of the system using MD simulations, many of ideas and hypothesis of experimentalists could be evaluated in this biophysicsbased model, before any experiments. Many of processing conditions and physical parameters of HPCD such as pressure, temperature, cosolvents, and additives can be examined by this method. It can predominately reduce the time and cost of the researches. For example, the effects of CO2-water mixing on the rate and performance of the process may be evaluated by MD simulation. Myoglobin is selected as model protein which there are some structural experimental evidences for that in HPCD process [46]. Moreover, lysozyme enzyme and chymotrypsin inhibitor 2 (CI2) protein were used to evaluate the simulation data. In the current work, biphasic systems including aqueous solutions of enzyme or protein and scCO2 is regarded as HPCD. However, HPCD also has been used for sterilization of solid materials such as heat sensitive bio-implants and the simulations could be extended to such materials in the future studies.

LINCS and SETTLE methods were applied for fixing bonds in the simulations [54,55]. Cut-off distance of 1.0 nm is considered for van der Waals and columbic interactions. Particle Mesh Ewald (PME) is applied as an algorithm for treating the long range electrostatic interactions [56]. Steepest-descent mathematical algorithm was used for energy minimization. The temperature and pressure were controlled by Berendsen algorithm [57]. 2.3. Simulation details Some sodium and chloride ions were added to each boxes for neutralization purpose. A simulation box was constructed with dimensions of 6.7☓6.7☓18 nm. Myoglobin was centered at the bottom half of the simulation box. The bottom half was filled by water and the above empty section was filled by pre-equilibrated CO2 phase. In the case of lysozyme and CI2 same conditions were used but with the different distances from the interface. After energy minimization of each box, the temperature and pressure were gradually increased to the experimental values in NVT and NPT ensembles, respectively. Initial Maxwell–Boltzmann velocities were generated randomly in NVT simulation and the system was equilibrated by gradually heating from 250 to 308 K over a 100 ps simulation. Afterwards, the pressure was gradually increased from 1 to 300 bar in series of NPT simulations with 100 ps equilibration time. In both NVT and NPT simulation, the heavy atoms of the systems were position restrained to relax the solvent molecules and better protein solvation. As the isothermal compressibility of CO2/water mixture become very similar to the pure state at the pressures higher than 10 MPa [58], we have assumed the compressibility is uniform through the system and its value is based on high pressure values of isothermal compressibility of pure states [58,59]. The convergence of force field parameters was evaluated in gmx energy module of GROMACS by testing the appropriate potential, temperature, and pressure in each simulation. The temperature and pressure were selected based on the experimental work of Ishikawa et al [46] and are 308 K and 30 MPa. After reaching to the desired temperature and pressure, system equilibration was performed in 200 ns simulation. To prevent any dependencies of the results on the initial conditions, production run was repeated ten times with the different initial orientation of protein toward z-axis. 2.4. Analysis All graphics were made by PyMOL molecular graphics software [60]. 3. Results and discussions To have unpredictable results, simulation parameters and conditions were assigned to be very similar to the experimental vessel of HPCD [4,15,21,46]. For this aim, protein is placed in the center of bottom half of a simulation box, which is filled with water up to the middle. Then, the above empty section of the box was charged with preequilibrated CO2 molecules until filling. Afterwards, the pressure was gradually increased to the appropriate amounts. Simulations were repeated 10 times with the different initial random orientations of protein toward z-axis of the simulation box. Time-dependent evolution of one representative simulation system is graphically depicted in Fig. 1. It can be seen that CO2 and water form a distinct biphasic system, which is consistent with the results of other CO2/water simulations [61,62]. Interestingly, the protein gradually migrates from pure aqueous phase toward the CO2/water interface and reaches to that in about 40 ns of MD simulation, showing significant conformational changes there. All of the ten simulations reproduced similar results, implying a very fast protein adsorption at CO2/water interface. As shown in Fig. 1, protein

2. Materials and methods 2.1. Software and parameters All MD simulations were performed by GROMACS package [47] version 2018 and GROMOS54a7 [48] force field. Myoglobin (pdb code:1mbn [49]), lysozyme (pdb code: 1lz1 [50]) and CI2 (pdb code: 1ypc [51]) were used as protein and enzyme models. The force field parameters of CO2 and water molecules are based on EPM2 [52] and SPC/E [53], respectively.

2

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

Fig. 1. Graphical representation of myoglobin during simulation in a HPCD system. Simulation is initiated from protein in the aqueous phase. Protein gradually transferred to the interface and remains there until final equilibration.

undergoes significant structural changes from globular to flat and extended conformation. These simulation results reveal that the protein can be inactivated by a denaturation mechanism and this denaturation probably occurs at the interface. The results are in agreement with the experimental observation of Ishikawa et al. [46] in which the myoglobin was treated by microbubbles of CO2 in the aqueous solutions. They have seen significant changes in the structure of myoglobin using circular dichroic (CD) experiments. The results of these investigators are helpful because they are the rare structural insights about protein in HPCD system. However, their results are not in situ observations. They performed their spectroscopic measurements after depressurization of CO2 and in mild conditions. They also do not provide a distinct structural mechanism based on their observations. Here, these simulations not only show the protein unfolding and denaturation in a HPCD system, but it also can depict a molecular level picture for this procedure. To our knowledge, these are the first structural insights, which show that the protein denaturation could be occurred at CO2/water interface in a HPCD pasteurization system. The phenomenon reported here for protein in CO2/water interface is similar to the more famous phenomena of protein adsorptions at air/ water [63,64] and oil/water [65,66] interfaces. Although there are various experimental [67–69] and theoretical [70–75] structural data about these processes, there is no similar structural evidences about the proteins and enzymes in CO2/water biphasic systems. Moreover, while protein denaturation at air/water and oil/water interfaces are almost accepted scientific issue, there is no direct structural evidence of protein denaturation at CO2/water interface. However, there are rare experimental reports, which support our simulation data by showing

possible interfacial properties of ovalbumin [76] and lysozyme [77] at CO2/water interface. These studies used pendant drop tensiometry [78] to show the interfacial properties with no structural data about the microstructure and conformational properties of proteins at the interface. Here, the structural properties of myoglobin were extensively examined based on its trajectory in the simulation. To have more insights, protein is also simulated in systems including only water or monophasic scCO2, considering the same conditions to the water/CO2 biphasic system. Root mean square deviation (rmsd) is a common statistical analysis for the trajectories of MD simulations. In protein simulation, it is an indicator of protein stability and tertiary structure intactness through comparison of the initial and the simulated structures. The values of rmsd for the protein during MD simulation of the different systems are demonstrated in Fig. 2(a). It can be seen that the protein has a normal rmsd (about 0.25 nm) in the pure aqueous phase. This shows that protein is remained in its native state in the aqueous conditions and any sign of instability cannot be seen during the simulation. In monophasic scCO2, rmsd has an initial sharp increment, which is continued with a normal termination at rmsd values about 0.45 nm. This result in in agreement with the previous MD simulations, showing the protein instability in monophasic scCO2 [37,38]. However, based on the values of rmsd, this instability on the myoglobin conformation is not as severe as it can be denatured. In the other hand, while rmsd values are in very normal ranges in the initial time of simulation in biphasic HPCD system, there is a sudden increment after arriving the protein at the interface with rmsd values of 1.5 nm. The 3D structures of myoglobin after simulation in different systems are shown in Fig. 2(b). This picture confirms the integrity of protein in water, 3

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

Fig. 2. (a) Rmsd values of myoglobin protein in different conditions, which were calculated, based on the α-carbons (b) Carton representations of myoglobin structure after simulation in different systems.

partial denaturation in scCO2, and complete decomposition and denaturation in HPCD system. The result of HPCD simulation shows that the tertiary structures of the protein are very different at the initial time of the simulation (in aqueous phase) and the equilibrated state (in contact with the CO2 interface). The simulation implies that the unfolding is occurred only at the interface region and not in the aqueous phase of HPCD. This is in contrast to the hypothesis of enzyme inactivation due to the dissolved and diffused CO2 in the aqueous phase. Therefore, the enzyme inactivation by lowering pH in the aqueous phase is declined by the same reason. As CO2 is regarded as hydrophobic fluid at high pressures, its effect on the internal hydrophobic residues while protein reaches to the interfacial region may be a key in the molecular mechanism of denaturation. To explore such effects, time-course changes of the myoglobin tertiary structure are graphically shown in Fig. 3 while green spheres highlight the amino acid side chains of the hydrophobic core and patches. Hydrophobic residues are surrounded by surface helixes, which prevent their direct contacts with the solvent molecules at the aqueous phase (Fig. 3, 2 ns). It can be seen that the hydrophobic regions at the protein cores were expanded during arrival to the interface and the protein expansion and unfolding were happened until the final of the simulation. These events show that the protein inactivation by HPCD process mainly occurs due to the weakening of the hydrophobic interactions in the protein tertiary structure. The helical contents of the protein which are demonstrated in Fig. 4(a) also show significant reduction in the secondary structure of myoglobin in agreement with the experiments of Ishikawa et al. [46]. However, about 50% of helixes remain native until the final of the simulation implying the possible interfacial position of the protein in HPCD system. Some of the helix structures can remain in the aqueous phase and show structural intactness. The dynamical behavior of the different residues during migration of the protein from the aqueous to the interface is an important factor, which can help to understand the

Fig. 3. Time-course changes of the tertiary structure of myoglobin. Green spheres highlight hydrophobic cores. At the first moments of the simulation hydrophobic residues were surrounded by surface helixes, while the come to the protein surface after arriving the protein at the CO2/water interface.

molecular mechanism. Root mean square fluctuation (rmsf) analysis shows the residues dynamics in common MD simulations of the proteins. Rmsf values were calculated for myoglobin sequence during the different simulation intervals (Fig. 4(b), 0–40 ns for the aqueous and 40–200 ns for the 4

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

Fig. 4. (a) The helical contents of myoglobin during MD simulation in HPCD system (b) Rmsf values for myoglobin residues. Protein residues show normal values of fluctuation before reaching to the interface (0–40 ns), while high fluctuating sequences are appeared at the interface (0–200 ns).

profiles of CO2 and water in the presence and absence of protein are shown in Fig. 6. The pbc conditions are considered while CO2 molecules form middle of the simulation system. It can be seen that the water density (blue line) is near to zero in the middle of box, which shows that water molecules are not present in CO2 phase (Fig. 6(a)). However, CO2 density is not zero about the two sides of CO2 phase, showing the diffusion of CO2 molecules in the aqueous phase. Density profiles of CO2 and water are slightly different while protein is dissolved in the aqueous phase of HPCD (Fig. 6(b)). Sharp reductions in water density about 7.5 nm from the center of system is due to the presence of protein. In this condition, CO2 density decreases in CO2 phase while increases in the aqueous phase about the protein. The protein situation in the aqueous phase was graphically depicted in Fig. 6(c) while removing one of the two phases in each section. As can be seen in the figure, the diffused CO2 molecules in the aqueous phase, which have a faster dynamics, make a hydrophobic microenvironment for protein extraction to the interface. In HPCD system, CO2 molecules diffuse to the aqueous phase about protein. These diffused molecules reduce water density about protein, make hydrophobic microenvironment, and ultimately lead to the protein adsorption to the CO2/water interface. To confirm the obtained results in the case of myoglobin protein, lysozyme enzyme is also simulated in HPCD system. Rmsd and conformations of lysozyme in HPCD system have shown in Fig. 7 based on the time of simulation and position of enzyme. Green spheres show side chains of the hydrophobic residues in the main hydrophobic core of lysozyme. As shown in the figure, the tertiary structure of lysozyme is significantly altered and the hydrophobic core is expanded after reaching the enzyme at interfacial region. These results imply that lysozyme enzyme can be deactivated by interfacial denaturation mechanism,

interface). At the aqueous phase, protein shows normal flexibility. However, it extremely fluctuates after lying at the interface. Rmsf values at the interface show a dual behavior and can be divided into two regions, some residues with faster and some with lower dynamics. This shows the dual dynamical behavior of the different residues after arrival of the protein to the interface. It seems that some polar amino acids that encounter with CO2 phase, due to the inappropriate interaction with nonpolar nature of the compressed CO2, return to the aqueous phase, leading to increasing the dynamics and flexibility in the surrounding sequences. Releasing the hydrophobic core amino acids to the CO2 phase also can establish similar fluctuating sequences. Fig. 5 graphically shows both of these phenomena in a clear way. Many of polar residues attempt to interact with the aqueous phase while hydrophobic non-polar residues escape from water to the CO2 phase. This is an excellent biophysical picture, which can clearly justify the interfacial properties of protein at CO2/water interface and the molecular mechanism of enzyme inactivation by HPCD. We have recently proposed a new molecular mechanism for the protein denaturation in monophasic scCO2 [43]. It was shown that lysine as a hydrophilic residue at the protein surface escapes from scCO2 solvent and forms non-native hydrogen bonds with other residues such as glutamic acids. These non-native interactions ultimately leads to the protein denaturant in scCO2. Here, we faced with the similar behavior of escaping the hydrophilic residues from CO2 phase. However, here, residues such as lysines prefer water molecules at the interface for forming the hydrogen bonds. This also leads to the protein denaturation. It seems that the mobility and diffusivity of CO2 molecules have an especial role in the adsorption of protein to the interface. The density 5

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

Fig. 5. The orientation of myoglobin and its residues at the interface. Only the surface residues are highlighted in the figure. Polar residues tend to be in the aqueous phase while non-polar residues are solvated by CO2 phase.

Fig. 6. The density profile of CO2 and water (a) in the absence of the protein and (b) in the presence of the protein. (c) Graphical representation of HPCD. CO2 molecules diffuse into the aqueous phase while water molecules do not exist in the CO2 phase.

surface of gas become higher and the distances of proteins and enzymes from interfacial regions become lower. To obtain detailed results about this effect, we have simulated the system of three CI2 proteins varying their distances from CO2/water interface. The results of this simulation are depicted in Fig. 8. Interestingly, the denaturation process is very distance-dependent. Closer protein to the interface shows a sharp increment during initial time (20 ns) of the simulation, which shows the early denaturation of this protein. The protein that has middle distance (4 nm) is denatured by showing high rmsd values after about 100 ns of

which confirms the obtained molecular mechanism for myoglobin denaturation. In the case of lysozyme simulation, we have reduced the distance from interphase in comparison with myoglobin in HPCD. This reduction makes the denaturation process of lysozyme occurs at shorter simulation time than that of myoglobin (Figs. 2(a) and 7). The distance from the interface can be related to the mixing effects and the size of CO2 bubbles in HPCD system. It was shown that using mico-bubbles of CO2 could significantly improve the enzyme deactivation process [6,8,16]. In other words, by micronizing the CO2 bubbles the accessible 6

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

Fig. 7. Rmsd values for lysozyme along with its carton presentation during simulation in HPCD system. At the initial time of simulation, lysozyme is in the aqueous phase and after arriving to the interface tertiary structure becomes decomposed.

Fig. 8. Distance-dependent rmsd of CI2 protein in HPCD system. Proteins were putted at the different distances from the CO2/water interface.

cases, pressurized CO2 is used for activating of enzymes, while it also can be used for inactivation purposes in the pasteurization process. Here, the processes of protein and enzyme inactivation by pressurized CO2 in the aqueous phase were simulated for the first time. Our results show that a proteins and enzymes can be unfolded by an “interfacial denaturation” mechanism. In agreement with the experimental observations, our calculations show distinct structural behaviors in a HPCD process. It was found that the tertiary structure of the proteins and enzymes is significantly changed from globular to a flat and extended conformation. Although the secondary structures also were reduced in HPCD process, half of that remained native until final simulation, showing an interfacial behavior of protein in this system. The main molecular mechanism of enzyme inactivation in HPCD includes

simulation. However, the protein with further distance from the interface is remained at lower rmsd values and it seems that cannot be denatured during 200 ns MD simulation. These results imply that if only one phase exists in HPCD system, inactivation of protein that are far from this interfacial region may be inefficient. Bubbling pressurized micro-size CO2 and also mixing the processing vessel make protein and enzyme to become accessible to the interfacial regions.

4. Conclusions Protein inactivation mechanism in high pressure CO2 pasteurization is poorly understood at the molecular levels. There are controversial opinions about systems including enzyme and CO2 molecules. In some 7

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

releasing the hydrophobic cores to the CO2 phase and escaping of the hydrophilic surface residues to the aqueous phase, which lead to protein denaturation. This work was essentially about the mechanistic aspects of the HPCD. However to have more insights for optimizing the HPCD process, the methodology should be improved. It was found that the applying of a confined space might be affected on the characteristics of the different molecular component of the simulation box [79]. This issue should be resolved in future works. The using of coarse-grained instead of all-atom force fields can help to reduce computational demands of such huge systems. In this way, simulation of more complicated biochemical systems may be possible. Moreover, the effects of the different variables such as temperature, pressure, protein concentration and CO2/water could be examined.

carbon dioxide microbubbles, J. Food Eng. 171 (2016) 52–56. [17] F. Gui, F. Chen, J. Wu, Z. Wang, X. Liao, X. Hu, Inactivation and structural change of horseradish peroxidase treated with supercritical carbon dioxide, Food Chem. 97 (3) (2006) 480–489. [18] K. Marszałek, P. Doesburg, S. Starzonek, J. Szczepańska, Ł. Woźniak, J.M. Lorenzo, S. Skąpska, S. Rzoska, F.J. Barba, Comparative effect of supercritical carbon dioxide and high pressure processing on structural changes and activity loss of oxidoreductive enzymes, J. Co2 Util. 29 (2019) 46–56. [19] F. Gui, J. Wu, F. Chen, X. Liao, X. Hu, Z. Zhang, Z. Wang, Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxide, Food Chem. 100 (4) (2007) 1678–1685. [20] L. Manzocco, S. Plazzotta, S. Spilimbergo, M.C. Nicoli, Impact of high-pressure carbon dioxide on polyphenoloxidase activity and stability of fresh apple juice, LWT - Food Sci. Technol. 85 (2017) 363–371. [21] T. Duong, M. Balaban, Optimisation of the process parameters of combined high hydrostatic pressure and dense phase carbon dioxide on enzyme inactivation in feijoa (Acca sellowiana) puree using response surface methodology, Innov. Food Sci. Emerg. Technol. 26 (2014) 93–101. [22] G. Ceni, M. Fernandes Silva, C. Valério Jr, R.L. Cansian, J.V. Oliveira, C. Dalla Rosa, M.A. Mazutti, Continuous inactivation of alkaline phosphatase and Escherichia coli in milk using compressed carbon dioxide as inactivating agent, J. CO2 Util. 13 (2016) 24–28. [23] A.J. Messiano, E.J. Beckman, A.J. Russell, C.R. 623., Supercritical biocatalysis, Chem. Rev. 99 (1999) 623–633. [24] T. Matsuda, K. Watanabe, T. Harada, K. Nakamura, Enzymatic reactions in supercritical CO2: carboxylation, asymmetric reduction and esterification, Catal. Today 96 (3) (2004) 103–111. [25] H.R. Hobbs, N.R. Thomas, Biocatalysis in supercritical fluids, in fluorous solvents, and under solvent-free conditions, Chem. Rev. 107 (2007) 2786–2820. [26] T. Matsuda, Recent progress in biocatalysis using supercritical carbon dioxide, J. Biosci. Bioeng. 115 (3) (2013) 233–241. [27] Y. Ikushima, N. Saito, M. Arai, H.W. Blanch, Activation of a lipase triggered by interactions with supercritical carbon dioxide in the near-critical region, J. Phys. Chem. 99 (22) (1995) 8941–8944. [28] N. Mase, T. Sako, Y. Horikawa, K. Takabe, Novel strategic lipase-catalyzed asymmetrization of 1,3-propanediacetate in supercritical carbon dioxide, Tetrahedron Lett. 44 (28) (2003) 5175–5178. [29] S. Kamat, G. Critchley, E.J. Beckman, A.J. Russell, Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: III. Does carbon dioxide covalently modify enzymes? Biotechnol. Bioeng. 46 (6) (1995) 610–620. [30] M. Habulin, Z. Knez, Activity and stability of lipases from different sources in supercritical carbon dioxide and near-critical propane, J. Cemical Technol. Biotechnol. 76 (2001) 1260–1266. [31] M. Karplus, J.A. McCammon, Molecular dynamics simulations of biomolecules, Nat. Struct. Biol. 9 (9) (2002) 646–652. [32] R.O. Dror, R.M. Dirks, J.P. Grossman, H. Xu, D.E. Shaw, Biomolecular simulation: a computational microscope for molecular biology, Annu. Rev. Biophys. 41 (1) (2012) 429–452. [33] V. Daggett, Molecular dynamics simulations of the protein unfolding/folding reaction, Acc. Chem. Res. 35 (6) (2002) 422–429. [34] X. Liu, D. Shi, S. Zhou, H. Liu, H. Liu, X. Yao, Molecular dynamics simulations and novel drug discovery, Expert Opin. Drug Discov. 13 (1) (2018) 23–37. [35] A. Singh, S.K. Vanga, V. Orsat, V. Raghavan, Application of molecular dynamic simulation to study food proteins: a review, Crit. Rev. Food Sci. Nutr. 58 (16) (2018) 2779–2789. [36] G. Chen, K. Huang, M. Miao, B. Feng, O.H. Campanella, Molecular dynamics simulation for mechanism elucidation of food processing and safety: state of the art, Compr. Rev. Food Sci. Food Saf. 18 (1) (2019) 243–263. [37] M.R. Housaindokht, M.R. Bozorgmehr, H. Monhemi, Structural behavior of Candida antarctica lipase B in water and supercritical carbon dioxide: a molecular dynamic simulation study, J. Supercrit. Fluid. 63 (2012) 180–186. [38] H. Monhemi, M.R. Housaindokht, How enzymes can remain active and stable in a compressed gas? New insights into the conformational stability of Candida antarctica lipase B in near-critical propane, J. Supercrit. Fluids 72 (2012) 161–167. [39] H. Monhemi, M.R. Housaindokht, M.R. Bozorgmehr, M.S.S. Googheri, Enzyme is stabilized by a protection layer of ionic liquids in supercritical CO2: insights from molecular dynamic simulation, J. Supercrit. Fluids 69 (2012) 1–7. [40] M.R. Housaindokht, H. Monhemi, The open lid conformation of the lipase is explored in the compressed gas: new insights from molecular dynamic simulation, J. Mol. Catal. B Enzym. 87 (0) (2013) 135–138. [41] H. Monhemi, M.R. Housaindokht, A. Nakhaei Pour, Effects of natural osmolytes on the protein structure in supercritical CO2: molecular level evidence, J. Phys. Chem. B 119 (33) (2015) 10406–10416. [42] H. Monhemi, M.R. Housaindokht, Chemical modification of biocatalyst for function in supercritical CO2: in silico redesign of stable lipase, J. Supercrit. Fluids 117 (2016) 147–163. [43] H. Monhemi, M.R. Housaindokht, The molecular mechanism of protein denaturation in supercritical CO2: the role of exposed lysine residues is explored, J. Supercrit. Fluids (2018). [44] H. Monhemi, M.R. Housaindokht, A.A. Moosavi-Movahedi, M.R. Bozorgmehr, How a protein can remain stable in a solvent with high content of urea: insights from molecular dynamics simulation of Candida antarctica lipase B in urea : choline chloride deep eutectic solvent, J. Chem. Soc. Faraday Trans. 16 (28) (2014) 14882–14893. [45] M.R. Housaindokht, H. Monhemi, H.E. Hosseini, M.S. Sadeghi Googheri, R.I. Najafabadi, N. Ashraf, M. Gholizadeh, It is explored that ionic liquids can be

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Acknowledgement This work is supported by research council of the University of Neyshabur. References [1] L. Garcia-Gonzalez, A.H. Geeraerd, S. Spilimbergo, K. Elst, L. Van Ginneken, J. Debevere, J.F. Van Impe, F. Devlieghere, High pressure carbon dioxide inactivation of microorganisms in foods: the past, the present and the future, Int. J. Food Microbiol. 117 (1) (2007) 1–28. [2] L. Manzocco, A. Ignat, F. Valoppi, K.R. Burrafato, G. Lippe, S. Spilimbergo, M.C. Nicoli, Inactivation of mushroom polyphenoloxidase in model systems exposed to high-pressure carbon dioxide, J. Supercrit. Fluids 107 (2016) 669–675. [3] L. Zhou, Y. Zhang, X. Hu, X. Liao, J. He, Comparison of the inactivation kinetics of pectin methylesterases from carrot and peach by high-pressure carbon dioxide, Food Chem. 115 (2) (2009) 449–455. [4] M.O. Balaban, A.G. Arreola, M. Marshall, A. Peplow, C.I. Wei, J. Cornell, Inactivation of pectinesterase in orange juice by supercritical carbon dioxide, J. Food Sci. 56 (3) (1991) 743–746. [5] L. Zhou, J. Wu, X. Hu, X. Zhi, X. Liao, Alterations in the activity and structure of pectin methylesterase treated by high pressure carbon dioxide, J. Agric. Food Chem. 57 (5) (2009) 1890–1895. [6] H. Ishikawa, M. Shimoda, T. Kawano, Y. Osajima, Inactivation of enzymes in an aqueous solution by micro-bubbles of supercritical carbon dioxide, Biosci. Biotechnol. Biochem. 59 (4) (1995) 628–631. [7] X. Liao, Y. Zhang, J. Bei, X. Hu, J. Wu, Alterations of molecular properties of lipoxygenase induced by dense phase carbon dioxide, Innov. Food Sci. Emerg. Technol. 10 (1) (2009) 47–53. [8] T. Yoshimura, M. Shimoda, H. Ishikawa, M. Miyake, I. Hayakawa, K. Matsumoto, Y. Osajima, Inactivation kinetics of enzymes by using continuous treatment with microbubbles of supercritical carbon dioxide, J. Food Sci. 66 (5) (2001) 694–697. [9] Ó. Benito-Román, M.T. Sanz, A.E. Illera, R. Melgosa, J.M. Benito, S. Beltrán, Pectin methylesterase inactivation by high pressure carbon dioxide (HPCD), J. Supercrit. Fluids 145 (2019) 111–121. [10] B.K. Tiwari, N. Brunton, C. Brennan, P.J. Cullen, C.P. O’Donnell, Application of supercritical carbon dioxide to fruit and vegetables: extraction, processing, and preservation AU - Rawson, A, Food Rev. Int. 28 (3) (2012) 253–276. [11] S. Fabroni, M. Amenta, N. Timpanaro, P. Rapisarda, Supercritical carbon dioxidetreated blood orange juice as a new product in the fresh fruit juice market, Innov. Food Sci. Emerg. Technol. 11 (3) (2010) 477–484. [12] S.S. Karajanagi, R. Yoganathan, R. Mammucari, H. Park, J. Cox, S.M. Zeitels, R. Langer, N.R. Foster, Application of a dense gas technique for sterilizing soft biomaterials, Biotechnol. Bioeng. 108 (7) (2011) 1716–1725. [13] A. Bernhardt, M. Wehrl, B. Paul, T. Hochmuth, M. Schumacher, K. Schütz, M. Gelinsky, Improved sterilization of sensitive biomaterials with supercritical carbon dioxide at low temperature, PLoS One 10 (6) (2015) e0129205-e0129205. [14] T. Iftikhar, M.E. Wagner, S.S.H. Rizvi, Enhanced inactivation of pectin methyl esterase in orange juice using modified supercritical carbon dioxide treatment, Int. J. Food Sci. Technol. 49 (3) (2014) 804–810. [15] H. Ishikawa, M. Shimoda, A. Yonekura, Y. Osajima, Inactivation of enzymes and decomposition of α-Helix structure by supercritical carbon dioxide microbubble method, J. Agric. Food Chem. 44 (9) (1996) 2646–2649. [16] F. Kobayashi, S. Odake, K. Kobayashi, H. Sakurai, Effect of pressure on the inactivation of enzymes and hiochi bacteria in unpasteurized sake by low-pressure

8

Journal of CO₂ Utilization xxx (xxxx) xxx–xxx

H. Monhemi and S. Dolatabadi

[46]

[47] [48]

[49] [50]

[51]

[52]

[53] [54] [55] [56] [57]

[58]

[59] [60] [61] [62]

[63]

suitable solvents for nitrile hydratase catalyzed reactions: a gift of the molecular modeling for the industry, J. Mol. Liq. 187 (2013) 30–42. H. Ishikawa, M. Shimoda, A. Yonekura, K. Mishima, K. Matsumoto, Y. Osajima, Irreversible unfolding of myoglobin in an aqueous solution by supercritical carbon dioxide, J. Agric. Food Chem. 48 (10) (2000) 4535–4539. E. Lindahl, B. Hess, D. van der Spoel, A message-passing parallel molecular dynamics implementation, Comput. Phys. Commun. 91 (1995) 43–56. N. Schmid, A.P. Eichenberger, A. Choutko, S. Riniker, M. Winger, A.E. Mark, W.F. van Gunsteren, Definition and testing of the GROMOS force-field versions 54A7 and 54B7, Eur. Biophys. J. 40 (7) (2011) 843. The Stereochemistry of the Protein Myoglobin, (2019) http://www.rcsb.org/ structure/1MBN. P.J. Artymiuk, C.C.F. Blake, Refinement of human lysozyme at 1.5 Å resolution analysis of non-bonded and hydrogen-bond interactions, J. Mol. Biol. 152 (4) (1981) 737–762. Y. Harpaz, N. Elmasry, A.R. Fersht, K. Henrick, Direct observation of better hydration at the N terminus of an alpha-helix with glycine rather than alanine as the N-cap residue, Proc. Natl. Acad. Sci. 91 (1) (1994) 311–315. J.G. Harris, K.H. Yung, Carbon dioxide’s liquid-vapor coexistence curve and critical properties as predicted by a simple molecular model, J. Phys. Chem. 99 (1995) 12021–12024. J. Hermans, H.J.C. Berendsen, W.F.V. Gunsteren, J.P.M. Postma, A consistent empirical potential for water–protein interactions, Biopolymers 23 (1984) 1513–1518. B. Hess, H. Bekker, H.J.C. Beredensen, J.E.M. Faraaije, LINCS: a linear constraint solver for molecular simulations, J. Comput. Chem. 18 (1997) 1463–1472. S. Miyamoto, P.A. Kollman, Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models, J. Comput. Chem. 13 (8) (1992) 952–962. T. Darden, D. York, L. Pedersen, Particle mesh Ewald: an Nlog(N) method for Ewald sums in large systems, J. Chem. Phys. 98 (1993) 10089–10092. H.J.C. Berendsen, J.P.M. Postma, W.F. van Gunsteren, A. DiNola, J.R. Haak, Molecular dynamics with coupling to an external bath, J. Chem. Phys. 81 (1984) 3684–3690. J. Zhang, X. Zhang, B. Han, J. He, Z. Liu, G. Yang, Study on intermolecular interactions in supercritical fluids by partial molar volume and isothermal compressibility, J. Supercrit. Fluids 22 (1) (2002) 15–19. R.A. Fine, F.J. Millero, Compressibility of water as a function of temperature and pressure, J. Chem. Phys. 59 (10) (1973) 5529–5536. W.L. Delano, The PyMOL Molecular Graphics System, citeulike-article-id:2816763 (2002) http://www.pymol.org. H. Zhang, S.J. Singer, Analysis of the subcritical carbon dioxide−water interface, J. Phys. Chem. A 115 (23) (2011) 6285–6296. L. Zhao, S. Lin, J.D. Mendenhall, P.K. Yuet, D. Blankschtein, Molecular dynamics investigation of the various atomic force contributions to the interfacial tension at the supercritical CO2–water interface, J. Phys. Chem. B 115 (19) (2011) 6076–6087. A.V. Makievski, V.B. Fainerman, M. Bree, R. Wüstneck, J. Krägel, R. Miller,

[64]

[65] [66] [67]

[68]

[69]

[70] [71] [72]

[73] [74]

[75] [76]

[77]

[78]

[79]

9

Adsorption of proteins at the liquid/air interface, J. Phys. Chem. B 102 (2) (1998) 417–425. H.H.J. de Jongh, H.A. Kosters, E. Kudryashova, M.B.J. Meinders, D. Trofimova, P.A. Wierenga, Protein adsorption at air–water interfaces: a combination of details, Biopolymers 74 (1‐2) (2004) 131–135. I. Langmuir, D.F. Waugh, The adsorption of proteins at oil-water interfaces and artificial protein-lipoid membranes, J. Gen. Physiol. 21 (6) (1938) 745–755. G. Yampolskaya, D. Platikanov, Proteins at fluid interfaces: adsorption layers and thin liquid films, Adv. Colloid Interface Sci. 128-130 (2006) 159–183. Y. Xiao, L. Konermann, Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry, Protein Sci. 24 (8) (2015) 1247–1256. E. D’Imprima, D. Floris, M. Joppe, R. Sánchez, M. Grininger, W. Kühlbrandt, The deadly touch: protein denaturation at the water-air interface and how to prevent it, bioRxiv (2018) 400432. Y.F. Yano, E. Arakawa, W. Voegeli, C. Kamezawa, T. Matsushita, Initial conformation of adsorbed proteins at an air–water interface, J. Phys. Chem. B 122 (17) (2018) 4662–4666. D.L. Cheung, Molecular simulation of hydrophobin adsorption at an oil–water interface, Langmuir 28 (23) (2012) 8730–8736. M. Cieplak, D.B. Allan, R.L. Leheny, D.H. Reich, Proteins at air–water interfaces: a coarse-grained model, Langmuir 30 (43) (2014) 12888–12896. D. Zare, K.M. McGrath, J.R. Allison, Deciphering β-Lactoglobulin interactions at an oil–water interface: a molecular dynamics study, Biomacromolecules 16 (6) (2015) 1855–1861. D.L. Cheung, Conformations of myoglobin-derived peptides at the air–water interface, Langmuir 32 (18) (2016) 4405–4414. D. Zare, J.R. Allison, K.M. McGrath, Molecular dynamics simulation of β-lactoglobulin at different oil/water interfaces, Biomacromolecules 17 (5) (2016) 1572–1581. Y. Zhao, M. Cieplak, Proteins at air–water and oil–water interfaces in an all-atom model, J. Chem. Soc. Faraday Trans. 19 (36) (2017) 25197–25206. F. Tewes, F. Boury, Effect of H2O−CO2 organization on ovalbumin adsorption at the supercritical CO2−water interface, J. Phys. Chem. B 109 (5) (2005) 1874–1881. G.D. Bothun, Y.W. Kho, J.A. Berberich, J.P. Shofner, T. Robertson, K.J. Tatum, B.L. Knutson, Surface activity of lysozyme and dipalmitoyl phosphatidylcholine vesicles at compressed and supercritical fluid interfaces, J. Phys. Chem. B 109 (51) (2005) 24495–24501. J.D. Berry, M.J. Neeson, R.R. Dagastine, D.Y.C. Chan, R.F. Tabor, Measurement of surface and interfacial tension using pendant drop tensiometry, J. Colloid Interface Sci. 454 (2015) 226–237. N. Zhang, J. Huo, B. Yang, X. Ruan, X. Zhang, J. Bao, W. Qi, G. He, Understanding of imidazolium group hydration and polymer structure for hydroxide anion conduction in hydrated imidazolium-g-PPO membrane by molecular dynamics simulations, Chem. Eng. Sci. 192 (2018) 1167–1176.