Variations in the enantioselectivity of salt-activated subtilisin induced by lyophilization

Including excess salt during lyophilization has been shown to increase the activity of freeze-dried subtilisin Carlsberg (SC) in anhydrous media by ov er 20,000-fold [Ru et al. (1999) Biotechnol Bioeng 63:233-241]. In the pres ent study, salt-activated SC (KCI-SC) showed a 30% enhancement in enantiose lectivity compared to the salt-free enzyme in a variety of organic solvents . Activity toward both enantiomers of N-acetyl-phenylalanine methyl ester ( APME) increased in tandem by 2-3 orders of magnitude in all solvents, indic ating that the mechanism of salt activation is inherent to the enzyme and d oes not strongly favor one enantiomer over the other. However, activity and enantioselectivity of salt-activated SC could be manipulated through chang es in the lyophilization conditions. Variations in lyophilization time, ini tial KCI concentration, and initial lyophilization volume altered enantiose lectivity over 2-fold. The changes in enantioselectivity reflected the acti vity for the L enantiomer, while the activity toward the D enantiomer was m ostly unaffected. The results indicate that the lyophilization time and fin al water content of the KCI-SC are important determinants of enzyme activit y for the L enantiomer, suggesting that the favored reaction is more sensit ive to the structural integrity of the salt-activated enzyme. (C) 2001 John Wiley & Sons, Inc.