Salts dramatically enhance activity of enzymes suspended in organic solvents

Khmelnitsky, Yuri L.; Welch, Stephanie H.; Clark, Douglas S.; Dordick, Jonathan S.

doi:10.1021/ja00085a066

Cited by 250 publications

(172 citation statements)

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“…To this end, Khmelnitsky et al (1994) devised a relatively simple strategy, which starts by dissolving the enzyme in a concentrated salt solution. Such conditions can lead to so-called preferential hydration (Timasheff 1993), whereby certain ions are excluded from the water near the protein's surface, and the protein is stabilized against unfolding.…”

Section: Dramatic Activation Of Enzymes In Nearly Dry Environmentsmentioning

confidence: 99%

“…Indeed, the salt-activation strategy was highly successful (Khmelnitsky et al 1994;Ru et al 1999Ru et al , 2000Ru et al , 2001Lindsay et al 2004). For example, when subtilisin Carlsberg was freeze-dried together with KCl, a dramatic activation effect was observed in several solvents, with a rate enhancement of nearly 4000-fold observed in hexane for a preparation containing 98% w/w KCl salt (Khmelnitsky et al 1994).…”

Section: Dramatic Activation Of Enzymes In Nearly Dry Environmentsmentioning

confidence: 99%

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Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

Clark

2004

Phil. Trans. R. Soc. Lond. B

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We have examined enzymes in nearly anhydrous organic solvents spanning a wide range of dielectric constants using a combination of electron paramagnetic resonance (EPR) spectroscopy, molecular dynamics simulations, high-pressure kinetic studies and the electrostatic model of Kirkwood. This approach enabled us to investigate the relationship between catalytic activity, protein flexibility and solvent polarity for an enzymatic reaction proceeding through a highly polar transition state in the near absence of water. Further insights into water-protein interactions and the involvement of water in enzyme structure and function have been obtained by EPR and multinuclear nuclear magnetic resonance studies of enzymes suspended and immobilized in organic solvents with and without added water. In these systems, correlations were observed between the water content and enzyme activity, flexibility, and active-site polarity, although the structural properties of suspended and immobilized enzymes differed markedly. These results have helped to elucidate the role of water in molecular events at the enzymic active site leading to improved biocatalysis in low-water environments.

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Section: Dramatic Activation Of Enzymes In Nearly Dry Environmentsmentioning

confidence: 99%

Section: Dramatic Activation Of Enzymes In Nearly Dry Environmentsmentioning

confidence: 99%

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

Clark

2004

Phil. Trans. R. Soc. Lond. B

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“…To counter this limitation, many methods have been introduced leading to improved enzyme activity in organic solvents. For example, control of the pH value (Yang et al, 1993), colyophilization with lyoprotectants (Dabulis and Klibanov, 1993) and salts (Khmelnitsky et al, 1994;Ru et al, 1999), addition of water-mimicking agents Kitaguchi et al, 1990), imprinting with substrates and substrate analogs (Rich and Dordick, 1997;Russell and Klibanov, 1988), immobilization (Orsat et al, 1994;Petro et al, 1996;Ruiz et al, 2000), solubilization Okahata et al, 1995a,b;Paradkar and Dordick, 1994;Wangikar et al, 1997;Xu et al, 1997), mutagenesis (Chen and Arnold, 1993), and solvent precipitation (Dai and Klibanov, 1999) represent methods that have been successful for improving the catalytic activity of enzymes. One of the most successful groups of activating additives identified thus far are macrocyclic compounds, which includes cyclodextrins Ooe et al, 1999;Santos et al, 1999) and crown ethers (Broos et al, 1995a;Engbersen et al, 1996;Itoh et al, 1996;Reinhoudt et al, 1989;van Unen, 2000;van Unen et al, 1998van Unen et al, , 2001.…”

Section: Introductionmentioning

confidence: 99%

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Santos

Vidal

Pacheco

et al. 2001

Biotech & Bioengineering

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Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (±)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V 0 , ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38°C and 56°C, respectively). No relationship between this property and V 0 was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V 0 was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of sub-tilisin activation by crown ethers, as recently suggested.

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“…Researchers have attempted to increase enzyme activity in organic media through a variety of techniques [PEGylation (19), ion-pairing (20), solid-state buffers (21)], one of the simplest being the inclusion of excipients during the lyophilization process (22)(23)(24)(25)(26)(27)(28). Of the various excipients used, including salts, sugars, and polymers, the most promising results have been obtained with salts.…”

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confidence: 99%

Water dynamics and salt-activation of enzymes in organic media: Mechanistic implications revealed by NMR spectroscopy

Eppler

Komor

Huynh

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg colyophilized with inorganic salts for activation in different organic solvents. Spectral editing was used to ensure that the relaxation times were associated with relatively mobile deuterons, which were contributed almost entirely by D2O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, ( c)D 2 O, in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (kcat͞KM)app of the biocatalyst preparation increased from 0.092 s ؊1 ⅐M ؊1 (saltfree) to 1,140 s ؊1 ⅐M ؊1 (98% CsF). A similar effect was apparent in acetone; the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% KF), with a corresponding increase in (kcat͞KM)app of 0.140 s ؊1 ⅐M ؊1 (salt-free) to 12.8 s ؊1 ⅐M ؊1 (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(kcat͞KM)app] and ( c)D 2 O were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (kcat͞KM)app and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzymebound water mobility mediated by the presence of salt act as a molecular lubricant and enhance enzyme flexibility in a manner functionally similar to temperature. Greater flexibility may permit a larger degree of local transition-state mobility, reflected by a more positive entropy of activation, for the salt-activated enzyme compared with the salt-free enzyme. This increased mobility may contribute to the dramatic increases in biocatalyst activity.enzyme activation ͉ organic solvents ͉ salts ͉ subtilisin Carlsberg I n recent years, the application of selective, nonhazardous biocatalysts for chemical synthesis has become an increasingly attractive alternative to traditional chemical methods. This trend is driven in part by the exquisite chemo-, regio-, and enantioselectivities commonly demonstrated by enzymes. The high demand for enantiomerically pure and selectively functionalized molecules, especially within the pharmaceutical industry, continues to spur the expanding interest in biocatalysis. Unfortunately, many compounds of interest to the pharmaceutical and related industries exhibit poor solubility and undergo deleterious side reactions (e.g., hydrolysis) in water; hence, they are not amenable to enzymatic reactions in conventional media.Nonaqueous biocatalysis, including enzymatic reactions in nearly anhydrous organic solvents, has emerged as an alternative approach to circumvent the limitations of aqueous-based reaction systems. There are drawbacks, however, to performing enzymatic reactions in organic solvents, most notably low biocatalytic activity (1-5). Much effort has been directed toward elucidating the mechanism(s) underlying the low activity exhibited by insoluble enzyme formulations in organic solvents. Poor com...

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Salts dramatically enhance activity of enzymes suspended in organic solvents

Cited by 250 publications

References 0 publications

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents

Water dynamics and salt-activation of enzymes in organic media: Mechanistic implications revealed by NMR spectroscopy

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