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Variations in the enantioselectivity of salt‐activated subtilisin induced by lyophilization
Author(s) -
Hsu WeiTing,
Clark Douglas S.
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.1055
Subject(s) - chemistry , subtilisin , enantiomer , salt (chemistry) , enzyme , anhydrous , phenylalanine , racemization , enzyme assay , racemic mixture , chromatography , stereochemistry , organic chemistry , biochemistry , amino acid
Including excess salt during lyophilization has been shown to increase the activity of freeze‐dried subtilisin Carlsberg (SC) in anhydrous media by over 20,000‐fold [Ru et al. (1999) Biotechnol Bioeng 63:233–241]. In the present study, salt‐activated SC (KCl‐SC) showed a 30% enhancement in enantioselectivity 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, indicating that the mechanism of salt activation is inherent to the enzyme and does not strongly favor one enantiomer over the other. However, activity and enantioselectivity of salt‐activated SC could be manipulated through changes in the lyophilization conditions. Variations in lyophilization time, initial KCl concentration, and initial lyophilization volume altered enantioselectivity over 2‐fold. The changes in enantioselectivity reflected the activity for the L enantiomer, while the activity toward the D enantiomer was mostly unaffected. The results indicate that the lyophilization time and final water content of the KCl‐SC are important determinants of enzyme activity for the L enantiomer, suggesting that the favored reaction is more sensitive to the structural integrity of the salt‐activated enzyme. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 231–237, 2001.