Am. Santos et al., Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents, BIOTECH BIO, 74(4), 2001, pp. 295-308
Colyophilization or codrying of subtilisin Carlsberg with the crown ethers
18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activi
ty in THF, acetonitrile, and 1,4-dioxane in the transesterification reactio
ns of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-
1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V-
o, ranged from less than 10-fold to more than 100-fold. All crown ethers ac
tivated subtilisin substantially, which excludes a specific macrocyclic eff
ect from being responsible. The secondary structure of subtilisin was studi
ed by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-cro
wn-5 led to a more nativelike structure of subtilisin in the organic solven
ts employed when compared with that of the dehydrated enzyme obtained from
buffer alone. However, the high level of activation with 12-crown-4 where t
his effect was not observed excluded overall structural preservation from b
eing the primary cause of the observed enzyme activation. The conformationa
l mobility of subtilisin was investigated by performing thermal denaturatio
n experiments in 1,4-dioxane. Although only a small effect of temperature o
n subtilisin structure was observed for the samples prepared with or withou
t 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature
at quite low temperatures (38 degreesC and 56 degreesC, respectively). No r
elationship between this property and V-o was evident, but increased confor
mational mobility of the protein decreased its storage stability. The possi
bility of a "molecular imprinting" effect was also tested by removing 18-cr
own-6 from the subtilisin-18-crown-6 colyophilizate by washing. V-o was onl
y halved as a result of this procedure, an effect insignificant compared wi
th the ca. 80-fold rate enhancement observed prior to washing in THF. This
suggests that molecular imprinting is likely the primary cause of subtilisi
n activation by crown ethers, as recently suggested. (C) 2001 John Wiley &
Sons, Inc.