Enzyme activation in organic solvents: Co‐lyophilization of subtilisin Carlsberg with methyl‐β‐cyclodextrin renders an enzyme catalyst more active than the cross‐linked enzyme crystals

Abstract

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.

Salt‐activation of nonhydrolase enzymes for use in organic solvents

“…Other investigators have added small hydrophobic molecules with the idea of protecting the hydrophobic pocket of the enzyme from collapsing (Dai and Klibanov, 1999). Recently, a few groups have shown that crown ethers, oligoamines, and cyclodextrins each are capable of protecting proteases (Broos et al, 1995;Hasegawa et al, 2003;Kudryashova et al, 2003;Montanez-Clemente et al, 2002;van Unen et al, 2002).…”

Use of high activity enzyme preparations in neat organic solvents for organic synthesis

“…vii. Enzymes crystallized and crosslinked [26][27][28]/enzymes crosslinked extensively after precipitation.…”

“…Pepin and Lortie (2001) [38] used CLEC prepared from CAL B for esterification of (R,S)-Ibuprofen with dodecanol in octane and found that just like Novozyme-435 (an immobilized form of Candida antarctica lipase B (CAL B) sold by Novozymes, Denmark), CLEC also gave best performance at a w =0.1. An interesting comparison with CLEC design has shown [27] that colyophilization of Subtilisin doi: 10.7243/2053-7670-1-1…”

Improved Enantioselectivity of Subtilisin Carlsberg towards Secondary Alcohols by Protein Engineering

“…The activity and enantioselectivity of an enzyme in organic solvent has been shown to depend on its structural flexibility and, thus, on the preparation technique and the reaction conditions (solvent and temperature) . Numerous preparatory methods have been shown to affect the enantioselectivity and activity of SC to various extents . However, the main bottleneck for efficient application of such stabilized SC preparations is low stability in organic solvent .…”