Substrate and water adsorption phenomena in a gas/solid enzymatic reactor

Dimoula, Kerasina; Pohl, Martina; Büchs, Jochen; Spieß, Antje C.

doi:10.1002/biot.200800291

Cited by 6 publications

(8 citation statements)

References 25 publications

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“…This decrease shows that salt (from suspension buffer before enzyme drying) as well as glycosylation strongly influence the water adsorption, as already observed with other proteins. [47] The shape of experimental water sorption and desorption curves were very similar to water sorption curves previously obtained for other proteins and followed the pattern of the BET model. [47][48][49] Moreover, the value of 0.063 mg of water per mg of dry protein found for BET monolayer water was in the range 0.05-0.07 found for other proteins.…”

Section: Discussionsupporting

confidence: 80%

“…[47] The shape of experimental water sorption and desorption curves were very similar to water sorption curves previously obtained for other proteins and followed the pattern of the BET model. [47][48][49] Moreover, the value of 0.063 mg of water per mg of dry protein found for BET monolayer water was in the range 0.05-0.07 found for other proteins. [48,49] The absence of hysteresis between adsorption and desorption indicated that there was no irreversibly bound water on the protein.…”

Section: Discussionsupporting

confidence: 80%

“…This hysteresis was mainly attributed to additives and leaching of biocatalyst away from the carrier surface, instead of being attributed to the protein itself. [47] When trying to find correlation between biocatalyst hydration state and CALB catalytic activity at different water activities in gas phase, it appears that the optimal water activity with respect to alcoholysis (a w = 0.05-0.1) and enantioselectivity (a w = 0.2) both correspond to a water activity level at which the water is highly structured. The overlaying phenomena of water adsorption and enzyme activity and selectivity will be investigated more precisely in the future.…”

Section: Discussionmentioning

confidence: 99%

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Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Branco¹,

Graber²,

Denis³

et al. 2009

ChemBioChem

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Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (a(w)) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein-bound water molecules increased linearly with a(w), until at a(w)=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate-binding site. At higher water activity, the thickness of the hydration shell increased up to 10 A close to a(w)=1. Above a limit of 1600 protein-bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate-binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

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Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Branco¹,

Graber²,

Denis³

et al. 2009

ChemBioChem

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show abstract

“…It allows in particular revealing the formation of a water monolayer and its weight, and the occurrence of free water for higher a w [17]. Yet, BET model is considered as a good mathematical tool to characterize the water adsorption isotherm and to determine the size of the monolayer for proteins, and this for a w up to 0.4.…”

Section: Water Adsorption By Immobilized Calb Studied By Water Adsormentioning

confidence: 99%

“…There have been very few experimental studies addressing both water and substrates adsorption on enzymes [17,18], although these adsorption phenomena might be a way to better elucidate enzyme kinetics and specificity. Recently we have studied molecular mechanism of hydration of free, deglycosylated, and desalted pure CALB in the gas phase.…”

Section: Introductionmentioning

confidence: 99%

Lipase hydration state in the gas phase: Sorption isotherm measurements and inverse gas chromatography

Márton

Chaput

Pierre

et al. 2010

Biotechnology Journal

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Fax : +33 5 46 45 82 65Abbreviations: IGC, Inverse Gas Chromatography a W , water thermodynamic activity, CALB, Candida antarctica Lipase B, BET, Brunauer-Emmet-Teller. AbstractThe adsorption of water and substrate on immobilized Candida antarctica lipase B was studied by performing adsorption isotherm measurements and using Inverse Gas Chromatography (IGC). Water adsorption isotherm of the immobilized enzyme showed singular profile absorption incompatible with the BET model, probably due to the hydrophobic nature of the support, leading to very low interactions with water. IGC allowed determining the evolution with a W of both dispersive surface energies and acidity and basicity constants of immobilized enzyme. These results showed that water molecules progressively covered immobilized enzyme, when increasing a W , leading to a saturation of polar groups above a W 0.1 and full coverage of the surface above a W 0.25. IGC also enabled relevant experiments to be performed to investigate the behavior of substrates under a W that they will experience, in a competitive situation with water. Results indicated that substrates had to displace water molecules in order to adsorb on the enzyme from a W values between 0.1 to 0.2, depending on the substrate. As the conditions used for these adsorption studies resemble the ones of the continuous enzymatic solid/gas reactor, in which activity and selectivity of the lipase were extensively studied, it was possible to link adsorption results with particular effects of water on enzyme properties.

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Effects of methanol on lipases: Molecular, kinetic and process issues in the production of biodiesel

Lotti

Pleiss

Valero

et al. 2014

Biotechnology Journal

158

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The biotechnological production of biodiesel is based on transesterification/esterification reactions between a source of fatty acids and a short-chain alcohol, usually methanol, catalysed by enzymes belonging to the class known as lipases. Several lipases used in industrial processes, although stable in the presence of other organic solvents, are inactivated by methanol at or below the concentration optimal for biodiesel production, making it necessary to use stepwise methanol feeding or pre-treatment of the enzyme. In this review article we focus on what is currently know about methanol inactivation of lipases, a phenomenon which is not common to all lipase enzymes, with the goal of improving the biocatalytic process. We suggest that different mechanisms can lead to inactivation of different lipases, in particular substrate inhibition and protein unfolding. Attempts to improve the performances of methanol sensitive lipases by mutagenesis as well as process engineering approaches are also summarized.

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Substrate and water adsorption phenomena in a gas/solid enzymatic reactor

Cited by 6 publications

References 25 publications

Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics Simulations

Lipase hydration state in the gas phase: Sorption isotherm measurements and inverse gas chromatography

Effects of methanol on lipases: Molecular, kinetic and process issues in the production of biodiesel

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