Improved Enzyme Activity and Enantioselectivity in Organic Solvents by Methyl-β-cyclodextrin

Griebenow, Kai; Laureano, Yanira Díaz; Santos, Angélica M.; Clemente, Ileana Montañez; Rodríguez, Luiz; Vidal, and Michael W.; Barletta, Gabriel

doi:10.1021/ja990515u

Cited by 111 publications

(85 citation statements)

References 52 publications

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“…Transesterification was performed, followed by high-performance liquid chromatography (HPLC), and analyzed as described by Griebenow et al (1999). The reaction was done with 100 mM N-acetyl-L-phenylalanine phenylester, 1 M 1-propanol, and 1 mg of subtilisin per milliliter of solvent using an orbital shaker (25°C and 250 rpm).…”

Section: Kinetic Measurementsmentioning

confidence: 99%

“…Transesterification of Vinylbutyrate with (±)-1-Phenylethanol (SecPhenethylalcohol)-After transesterification, gas chromatography was performed and analyzed as described by Griebenow et al (1999). The reaction was done with 70 mM (±)-1-phenylethanol, 100 mM vinylbutyrate, and 1 mg of subtilisin per milliliter of solvent on an orbital shaker (45°C and 300 rpm).…”

Section: Kinetic Measurementsmentioning

confidence: 99%

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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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Section: Kinetic Measurementsmentioning

confidence: 99%

Section: Kinetic Measurementsmentioning

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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“…Unfortunately, native enzymes typically exhibit very low activities in nonnative solvent environments (10)(11)(12)(13), although techniques have been developed to increase the activity of some enzymes to near aqueous levels (14)(15)(16)(17)(18). One such activation methodology, known as salt activation, is unique in that enzyme activity can be controlled over an unprecedented range (17,18) (i.e., several orders of magnitude) without modifying the amino acid sequence, temperature, or bulk solvent environment.…”

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confidence: 99%

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

Eppler

Hudson

Chase

et al. 2008

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

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Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, 1 H NMR relaxation, and 19 F NMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive ⌬⌬S † , for the more active biocatalyst preparations in organic solvents. In contrast, ⌬⌬H † was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and 1 H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal magnetization exchange, revealed that the enzyme's turnover number (k cat) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the millisecond regime, and uncorrelated with protein motions on the piconanosecond timescale. In addition, 19 F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity.enzyme activation ͉ enzyme dynamics ͉ NMR spectroscopy ͉ organic solvents ͉ subtilisin Carlsberg T he direct coupling of protein dynamics to enzymatic catalysis is strongly supported by recent studies of enzymes in aqueous solution (1-3). These studies have revealed insights into the connection between enzyme activity and protein dynamics [e.g., conformation exchange in CypA (4), Aquifex Adk lid movement (5), Met-20 loop dynamics in DHFR (6), loop motions leading to conformational change in RNase A (7), and modulation of surfaceglycosylated chymotrypsin structural dynamics and activity (8)], yet the relatively small range of enzyme activities accessible under aqueous conditions, without the use of protein modifications or alteration of the solvent, hinders thorough investigation of the central hypothesis that enzyme catalysis is intimately coupled to protein dynamics. One strategy for broadening the range of activities over which enzyme catalysis and protein dynamics can be studied concurrently is to employ nonaqueous or organic solvents. For example, a correlation between molecular dynamics, as evidenced by a reduced rate of amide H/D exchange, and biocatalyst activity has been observed for PEGylated subtilisin Carlsberg (SC) in 1,4-dioxane (9).Nonaqueous biocata...

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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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Improved Enzyme Activity and Enantioselectivity in Organic Solvents by Methyl-β-cyclodextrin

Cited by 111 publications

References 52 publications

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

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

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions

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

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