Rational design of thermostable carbonic anhydrase mutants using molecular dynamics simulations

The stability of enzymes is critical for their application in industrial processes, which generally require different conditions from the natural enzyme environment. Both rational and random protein engineering approaches have been used to increase stability, with the latter requiring extensive experimental effort for the screening of variants. Moreover, some general rules addressing the molecular origin of protein thermostability have been established. Herein, we demonstrate the use of molecular dynamics simulations to gain molecular level understanding of protein thermostability and to engineer stabilizing mutations. Carbonic anhydrase (CA) is an enzyme with a high potential for biotechnological carbon capture applications, provided it can be engineered to withstand the high temperature process environments, inevitable in most gas treatment units. In this study, we used molecular dynamics simulations at 343, 353, and 363 K to study the relationship between structure flexibility and thermostability in bacterial α-CAs and applied this knowledge to the design of mutants with increased stability. The most thermostable α-CA known, TaCA from Thermovibrio ammonificans, had the most rigid structure during molecular dynamics simulations, but also showed regions with high flexibility. The most flexible amino acids in these regions were identified from root mean square fluctuation (RMSF) studies, and stabilizing point mutations were predicted based on their capacity to improve the calculated free energy of unfolding. Disulfide bonds were also designed at sites with suitable geometries and selected based on their location at flexible sites, assessed by B-factor calculation. Molecular dynamics simulations allowed the identification of five mutants with lower RMSF of the overall structure at 400 K, compared to wild-type TaCA. Comparison of free-energy landscapes between wild-type TaCA and the most promising mutants, Pro165Cys–Gln170Cys and Asn140Gly, showed an increased conformational stability of the mutants at 400 K

Engineering of Thermovibrio ammonificans carbonic anhydrase mutants with increased thermostability

Carbonic anhydrase can be used as an additive to improve the efficiency of carbon capture and utilisation processes, due to its ability to increase the rate of CO2 absorption into solvents. Successful industrial application requires robust carbonic anhydrases, able to withstand process conditions and to perform consistently over long periods of time. Tolerance of high temperatures, pH and salt concentrations are particularly desirable features. We have previously used molecular dynamics simulations to rationally design four mutants of Thermovibrio ammonificans carbonic anhydrase with increased rigidity, and we hypothesized that this will result in an increased thermostability. Herein, we report on the successful recombinant expression and characterization of these mutants. Four of the TaCA variants showed increased stability at 90 ᵒC during 1 h, compared to wild-type. Two out of the four mutations predicted by the theoretical studies resulted in marked stabilization of the protein, with up to 3-fold higher time of half-life for mutant N140 G compared to the wild-type enzyme at 60 ᵒC. A significantly 50-fold increased ester hydrolysis activity was also observed with the most thermostable variant at 95 ᵒC compared to 25 ᵒC, suggesting an increased flexibility of the active site at high temperatures

Molecular differentiated initiator reactivity in the synthesis of poly(caprolactone)-based hydrophobic homopolymer and amphiphilic core corona star polymers

Macromolecules that possess three-dimensional, branched molecular structures are of great interest because they exhibit significantly differentiated application performance compared to conventional linear (straight chain) polymers. This paper reports the synthesis of 3- and 4-arm star branched polymers via ring opening polymerisation (ROP) utilising multi-functional hydroxyl initiators and Sn(Oct)2 as precatalyst. The structures produced include mono-functional hydrophobic and multi-functional amphiphilic core corona stars. The characteristics of the synthetic process were shown to be principally dependent upon the physical/dielectric properties of the initiators used. ROP’s using initiators that were more available to become directly involved with the Sn(Oct)2 in the “in-situ” formation of the true catalytic species were observed to require shorter reaction times. Use of microwave heating (MWH) in homopolymer star synthesis reduced reaction times compared to conventional heating (CH) equivalents, this was attributed to an increased rate of “in-situ” catalyst formation. However, in amphiphilic core corona star formation, the MWH polymerisations exhibited slower propagation rates than CH equivalents. This was attributed to macro-structuring within the reaction medium, which reduced the potential for reaction. It was concluded that CH experiments were less affected by this macro-structuring because it was disrupted by the thermal currents/gradients caused by the conductive/convective heating mechanisms. These gradients are much reduced/absent with MWH because it selectively heats specific species simultaneously throughout the entire volume of the reaction medium. These partitioning problems were overcome by introducing additional quantities of the species that had been determined to selectively heat

Numerical studies of shear-thinning droplet formation in a microfluidic T-junction using two-phase level-set method

A conservative level-set method (LSM) embedded in a computational fluid dynamics (CFD) simulation provides a useful approach for the studying the physics and underlying mechanism in two-phase flow. Detailed two-dimensional (2D) computational microfluidics flow simulations have been carried out to examine systematically the influence of different controlling parameters such as flow rates, viscosities, surface wettability, and interfacial tensions between two immiscible fluids on the non-Newtonian shear-thinning microdroplets generation process. For the two-phase flow system that neglects the Marangoni effect, the breakup process of shear-thinning microdroplets in cross-flowing immiscible liquids in a microfluidic device with a T-shaped geometry was predicted. Data for the rheological and physical properties of fluids obeying Carreau-Yasuda stress model were empirically obtained to support the computational work. The simulation results show that the relevant control parameters mentioned above have a strong impact on the size of shear-thinning droplets generated. Present computational studies on the role and relative importance of controlling parameters can be established as a conceptual framework of the non-Newtonian droplet generation process and relevant phenomena for future studies

Supercritical fluid chemistry : optimisation and miniaturisation

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Continuous-flow Heck synthesis of 4-methoxybiphenyl and methyl 4-methoxycinnamate in supercritical carbon dioxide expanded solvent solutions

The palladium metal catalysed Heck reaction of 4-iodoanisole with styrene or methyl acrylate has been studied in a continuous plug flow reactor (PFR) using supercritical carbon dioxide (scCO2) as the solvent, with THF and methanol as modifiers. The catalyst was 2% palladium on silica and the base was diisopropylethylamine due to its solubility in the reaction solvent. No phosphine co-catalysts were used so the work-up procedure was simplified and the green credentials of the reaction were enhanced. The reactions were studied as a function of temperature, pressure and flow rate and in the case of the reaction with styrene compared against a standard, stirred autoclave reaction. Conversion was determined and, in the case of the reaction with styrene, the isomeric product distribution was monitored by GC. In the case of the reaction with methyl acrylate the reactor was scaled from a 1.0 mm to 3.9 mm internal diameter and the conversion and turnover frequency determined. The results show that the Heck reaction can be effectively performed in scCO2 under continuous flow conditions with a palladium metal, phosphine-free catalyst, but care must be taken when selecting the reaction temperature in order to ensure the appropriate isomer distribution is achieved. Higher reaction temperatures were found to enhance formation of the branched terminal alkene isomer as opposed to the linear trans-isomer

Characterizing droplet breakup rates of shear-thinning dispersed phase in microreactors

A two-phase flow predictive model with the integration of conservative level-set method (LSM) and Carreau-Yasuda constitutive equation was developed herein. The LSM was chosen as a potential interface capturing scheme for elucidating the interfacial phenomena including insight into the mechanism of shear-thinning droplets. In present paper, the dynamics of shear-dependent droplet emergence, growth, detachment and translocation in a Newtonian microsystem were examined via computational fluid dynamics (CFD) analysis. Dilute sodium carboxymethylcellulose (Na-CMC) solution was treated as dispersed phase (70 mPa.s < ηo  C*˜0.40 wt%). This striking behaviour highlights the importance of rheological effects in flows with a shear-dependent fluid under various flow conditions. The viscous effect of Na-CMC fluids substantially affects the manipulation over the droplet pinch-off time and production rate. Thus, it necessitate the control of the shear rate by adjusting the flow conditions and aspect ratio of microchannels

Numerical Simulation of the Effect of Rheological Parameters on Shear-Thinning Droplet Formation

Immiscible non-Newtonian-Newtonian fluid systems in microfluidics constitute an essential study as non-Newtonian fluids consistently met in medical and biological systems. Although a large number of experimental investigations have been reported in this area, attempts to develop predictive models appear to be limited. This paper is an attempt to incorporate a non-Newtonian stress model together with front-tracking scheme used in computational fluid dynamics. A conservative two-phase level set method (LSM) was applied for capturing the droplet breakup dynamics and relevant hydrodynamics of shear-thinning carboxymethylcellulose (CMC) droplets. Our droplets comprise of 0.02wt% to 1.2wt% CMC solutions in a Newtonian continuous fluids system (olive oil) employed in a T-shaped microfluidic cell. A Carreau-Yasuda viscosity model for shear-thinning CMC droplets has been implemented. This shear-dependent constitutive model fitted well to our steady state non-linear shear measurements for polymeric CMC solutions, with asymptotic viscosities at zero and infinite shear rates, and with different degrees of shear thinning (η0/η∞) in steady state. The particular focus of this study was to systematically undergo parametric studies on the influence of rheological parameters of the specified model such as zero (η0) and infinite shear viscosity (η∞), and relaxation time (λ) on the droplet formation processes. The level set simulation predicted that the droplet diameter increases with increasing η0/η∞. The effect of η0/η∞ has been found to have more prominent impact on droplet diameter for higher CMC concentrations. The variation in droplet diameter becomes less significant at the higher degrees of shear-thinning for all concentrations of CMC dispersed solutions. In the limit of zero shear-thinning effect, the droplet diameter increases when the dispersed phase viscosity decreases. Additionally, the effect of λ on the droplet diameter is also discussed. The reciprocal of the characteristic relaxation time (1/λ) corresponds to a critical shear rate that indicates the onset shear rate for shear-thinning. As λ increases, the numerical studies clearly reveal that the droplet diameter is increasing until it reaches a plateau for larger values of λ. The influence of λ leads to a more significant impact on droplet diameter for higher CMC concentration. These findings will ultimately help in understanding the sensitivity of rheological parameters to the microdroplet formation.</jats:p