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Michels PC, Clark DS: Pressure-enhanced activity and stability of a hyperthermophilic deep-sea protease. Appl Environ Microbiol 1997, 63:3985-3991. A protease from Methanococcus jannaschii was partially purified and found to show increased enzyme activity up to 116°C, with activity still measurable at 130°C, which gives this enzyme one of the highest temperatures for activity reported. In addition, the activity and stability of the enzyme at high temperatures was increased upon increasing the pressure: raising the pressure to 500 atm at 125°C resulted in a 3.4- and 2.7-fold increase in activity and stability, respectively. Electron paramagnetic resonance spectroscopy of a suitably spin-labeled active-site serine indicated that the active-site geometry of the M, jannaschii protease is not substantially different from that of related mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures.
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Schmitke JL, Stern LJ, Klibanov AM: The crystal structure of subtilisin carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile, Proc Natl Acad Sci USA 1997, 94:4250-4255. The X-ray structure of subtilisin Carlsberg was determined to 2.6 Å resolution following replacement of water (the solvent used in crystallization) with anhydrous dioxane. Seven enzyme-bound dioxane molecules could be detected, and the locations of these molecules were distinct from enzyme-bound solvent in water or acetonitrile. Nevertheless, the overall structure of subtilisin in dioxane was essentially the same as in water or acetonitrile, indicating that the enzyme retains its bulk conformation even in vastly different media.
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Okahata Y, Mon T: Lipid coated enzymes as efficient catalysts in organic media. Trends Biotechnol 1997, 15:50-54. Complexes formed via precipitation of proteins with lipids in an aqueous solution have been prepared with lipases, β-galactosidase, and catalytic antibodies for solubilization in benzene, ethyl acetate, isooctane, isopropyl ether, dimethyl sulfoxide, and ethanol. In all cases, the native structure is presumed to exist and this gives rise to relatively high activity when the biocatalysts are compared to their insoluble nonderivatized counterparts in the aforementioned solvents.
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Wangikar PP, Michels PC, Clark DS, Dordick JS: Structure and function of subtilisin BPN solubilized in organic solvents. J Am Chem Soc 1997, 119:70-76. Subtilisin BPN soluble in organic solvent retained both native secondary and tertiary structure in octane and other nonpolar solvents. Conversely, rapid denaturation occurred in tetrahydrofuran (THF) and other polar organic solvents. Interestingly, partially denatured subtilisin in THF could be renatured in anhydrous octane, thereby providing the first demonstration of enzyme renaturation in a bulk organic solvent.
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Wang P, Sergeeva MV, Lim L, Dordick JS: Biocatalytic plastics as active and stable materials for biotransformations. Nat Biotechnol 1997, 15:789-793. Enzyme-containing polymeric materials have been prepared that show high levels of activity and stability in both aqueous and organic media, These 'biocatalytic plastics', containing α-chymotrypsin and subtilisin Carlsberg, consist of up to =50% (w/w) total protein in plastic materials such as poly(methyl methacrylate, polystyrene, poly(vinyl acetate), and poly(ethyl vinyl ether). One significant attribute of these biocatalytic plastics is their high level of stability in polar organic solvents, which in turn is at least partly manifested in higher observed activity, For example, rate enhancements of over four orders of magnitude over native containing α-chymotrypsin have been observed for the synthesis of a tyrosine-leucine dipeptide derivative. Other polar solvents such as ethyl acetate and acetonitrile also support peptide synthesis to a far greater degree than native α-chymotrypsin.
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Rich JO, Dordick JS: Controlling subtilisin activity and selectivity in organic media by imprinting with nucleophilic substrates. J Am Chem Soc 1997, 119:3245-3252. The activity and substrate specificity of subtilisin-catalyzed acylation of nucleosides in organic solvents can be controlled by lyophilizing the enzyme from an aqueous solution containing the substrate. This molecular imprinting technique was examined using thymidine as a model nucleoside, and the resulting subtilisin preparation was up to 50-fold more reactive toward thymidine acylation in nearly anhydrous tetrahydrofuran than subtilisin lyophilized from aqueous buffer in the absence of the nucleoside. Furthermore, it was possible to alter the substrate selectivity of subtilisin by lyophilizing the enzyme in the presence of a different nucleophilic substrate. For example, imprinting made possible the discrimination between structurally different nucleophiles, Imprinting was performed with thymidine and sucrose separately. The two imprinted enzyme preparations were then examined for both sucrose and thymidine acylation. For sucrose acylation, the sucrose-activated and thymidine-activated enzymes were 40-fold and fourfold more active, respectively, than the native enzyme, The trend was reversed, however, for thymidine acylation where the sucrose-enzyme and the thymidine-enzyme were ninefold and 52-fold more active, respectively, than the native enzyme.
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