Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea.
Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.
Guo
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Department of Chemical Engineering, 201 Gilman Hall, University of California, 94720-1462, Berkeley, CA, USA.
Clark
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D000096:Acetone / Q000737:chemistry; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000187:drug effects; D002384:Catalysis / Q000187:drug effects; D002918:Chymotrypsin / Q000737:chemistry* / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005612:Freeze Drying; D005663:Furans / Q000737:chemistry; D006586:Hexanes / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012997:Solvents / Q000737:chemistry*; D013379:Substrate Specificity / Q000187:drug effects; D013381:Subtilisins / Q000737:chemistry* / Q000187:drug effects; D014508:Urea / Q000737:chemistry* / Q000494:pharmacology
D005663:Furans; D006586:Hexanes; D012997:Solvents; D000096:Acetone; C018674:tetrahydrofuran; D014508:Urea; D000426:Alcohol Dehydrogenase; D006735:Horseradish Peroxidase; D013381:Subtilisins; D002918:Chymotrypsin; C117608:alpha-chymotrypsin