Cutinase activity and enantioselectivity in supercritical fluids

Fontes, Nuno; Almeida, M. Conceição; García, Silvia; Peres, Célia; Grave, J. J. Salverda de; Aires-Barros, M. Raquel; Soares, Cláudio M.; Maycock, Christopher D.; Barreiros, Susana

doi:10.1016/s0921-0423(98)80072-5

Cited by 3 publications

(6 citation statements)

References 15 publications

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“…sc±CO 2 is perhaps the most popular sc¯uid, but is not the best solvent from the standpoint of biocatalysis. Catalytic activities in this solvent usually are one order of magnitude lower when compared to other sc¯uids Borges de Carvalho et al, 1996;Fontes et al, 1998;Kamat et al, 1995;Marty et al, 1992;Mesiano et al, 1999). There are negative direct eects, such as CO 2 condensation with amine side chains, resulting in carbamate formation (Kamat et al, 1995).…”

Section: Zeolite Acid±base Effects Explain Intriguing Results In Sc Fmentioning

confidence: 99%

“…a w was normally controlled by salt hydrate pairs (500 mg of each form) in situ, namely Na 2 HPO 4 .2/0 (a w = 0.25) and Na 2 HPO 4 .7/2 (a w = 0.75) (Zacharis et al, 1997a). In the experiments reported in Figures 1 and 2, a w was not controlled by salt hydrates, and its value was calculated by dividing the water concentration in the reaction mixture by the water concentration in the same mixture at saturation (Fontes et al, 1998). The enzyme was always allowed to equilibrate with any solid components present (salt hydrate pair, zeolite, solid buer) in the bulk nonaqueous phase (solvent and substrate alcohol) for 2 h to allow ample time for water and ionic transfer.…”

Section: Apparatus and Experimental Techniquementioning

confidence: 99%

“…The fact that zeolites are themselves cation exchangers has been largely neglected in most studies reporting the use of these materials, many of which involve the use of zeolites as supports for enzyme immobilization (Fontes et al, 1998;Serralha et al, 1998;Xing et al, 2000). In a recent study (Serralha et al, 1998); however, the in¯uence of the structure and composition of a zeolite support on the catalytic activity of an enzyme has been reported.…”

Section: Introductionmentioning

confidence: 99%

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Zeolite molecular sieves have dramatic acid–base effects on enzymes in nonaqueous media

Fontes

Partridge

Halling

et al. 2001

Biotech & Bioengineering

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Zeolite molecular sieves very commonly are used as in situ drying agents in reaction mixtures of enzymes in nonaqueous media. They often affect enzyme behavior, and this has been interpreted in terms of altered hydration. Here, we show that zeolites can also have dramatic acid-base effects on enzymes in low water media, resulting from their cation-exchange ability. Initial rates of transesterification catalyzed by cross-linked crystals of subtilisin were compared in supercritical ethane, hexane, and acetonitrile with water activity fixed by pre-equilibration. Addition of zeolite NaA (4 A powder) still caused remarkable rate enhancements (up to 20-fold), despite the separate control of hydration. In the presence of excess of an alternative solid-state acid-base buffer, however, zeolite addition had no effect. The more commonly used Merck molecular sieves (type 3 A beads) had similar but somewhat smaller effects. All zeolites have ion-exchange ability and can exchange H+ for cations such as Na+ and K+. These exchanges will tend to affect the protonation state of acidic groups in the protein and, hence, enzymatic activity. Zeolites pre-equilibrated in aqueous suspensions of varying pH-pNa gave very different enzyme activities. Their differing basicities were demonstrated directly by equilibration with an indicator dissolved in toluene. The potential of zeolites as acid-base buffers for low-water media is discussed, and their ability to overcome pH memory is demonstrated.

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Section: Zeolite Acid±base Effects Explain Intriguing Results In Sc Fmentioning

confidence: 99%

Section: Apparatus and Experimental Techniquementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Zeolite molecular sieves have dramatic acid–base effects on enzymes in nonaqueous media

Fontes

Partridge

Halling

et al. 2001

Biotech & Bioengineering

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confidence: 99%

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

Micaêlo

Soares

2007

The FEBS Journal

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The ability of enzymes to work in media other than water is now widely accepted, and is the basis of extensive basic research on enzyme catalysis and many biotechnological applications [1]. The fact that most enzymes have evolved in an aqueous environment in living cells does not mean that they cannot be transferred and be functional in a completely different kind of medium [2][3][4]. Our recent molecular modeling studies have depicted the molecular mechanism of the effects of different hydration percentages on the structural [5] and enantioselective properties of enzymes [6] when placed in organic solvents such as hexane. Many experimental studies in the field of nonaqueous enzymology have focused their attention on demonstrating that the amount of water in the organic medium plays an important role in controlling the catalytic properties of the enzymes [5][6][7][8][9]. These studies have shown that when enzymes are used in organic solvents, water retains its fundamental role in controlling the physical properties of the enzyme, and this role probably cannot be taken by other solvent. In such systems, the effect of water is complicated to investigate, because this solvent is distributed in several phases; it can be in the vapor phase, adsorbed to the support material, dissolved in the organic liquid phase, or bound to the enzyme [10].Of the total water added to the organic medium, the effect of the organic solvents on the enzyme seems to be primarily due to the water that is bound to the enzyme [7,11]. This bound water is usually measured experimentally in terms of the thermodynamic activity of water, assuming that, for enzymatic reactions A comprehensive study of the hydration mechanism of an enzyme in nonaqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with 12 different hydration levels ranging from 5% to 100% (w ⁄ w) (weight of water ⁄ weight of protein). The ability of organic solvents to 'strip off' water from the enzyme surface was clearly dependent on the nature of the organic solvent. The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used. It was also shown that organic solvents determine the structure and dynamics of water at the enzyme surface. Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface. Ions seem to play an important role in the stabilization of exposed charged residues, mainly at low hydration levels. A common feature is found for the preferential localization of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surfac...

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“…These substrates are commonly used in transesterification reactions with vinyl butyrate carried out by serine proteases in nonaqueous solvents. The availability of experimental data for this reaction using cutinase in acetonitrile (40) and supercritical CO 2 (41) has prompted this choice. The model compound modeled in the enzyme active site was the second TI in the reaction mechanism of transesterification of sec-alcohols catalyzed by serine proteases (Fig.…”

Section: Substrate Modelingmentioning

confidence: 99%

Water Dependent Properties of Cutinase in Nonaqueous Solvents: A Computational Study of Enantioselectivity

Micaêlo

Teixeira

Baptista

et al. 2005

Biophysical Journal

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The catalytic properties of enzymes in nonaqueous solvents are known to be dependent on the nature of the solvent. Here we present a molecular modeling study of the enantioselective properties of the enzyme cutinase in hexane under varying hydration conditions. Previous simulation studies have shown that for this model enzyme in hexane, the structural and dynamical properties are affected by the amount of water associated with the protein, being more similar to the aqueous simulation at 5-10% of water content. The implications of the hydration levels on the enzyme resolution of (R,S)-1-phenylethanol and (R,S)-2-phenyl-1-propanol are investigated using free energy calculations of the tetrahedral intermediate (TI) model. With this model system we show that the enzyme enantioselective properties are under the control of the amount of water present in the organic media. Maximum enantioselectivity is achieved at 10% water content. The stabilizing effects of the catalytic histidine on the TI are evaluated at different water contents and shown to be correlated. The correlation between the amount of water present in the media and the structural, dynamical, and thermodynamic properties of the enzyme are examined as well as the active site discriminative power.

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Cutinase activity and enantioselectivity in supercritical fluids

Cited by 3 publications

References 15 publications

Zeolite molecular sieves have dramatic acid–base effects on enzymes in nonaqueous media

Zeolite molecular sieves have dramatic acid–base effects on enzymes in nonaqueous media

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

Water Dependent Properties of Cutinase in Nonaqueous Solvents: A Computational Study of Enantioselectivity

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