ABSTRACT
Scheme 13.1 CAL-B-catalyzed aldol addition of hexanal.The mutants Ser105Ala and Ser105Gly, which lack the nucleophilic serine residue in the catalytic triad, showed a clear aldolase activity. The specific activity of mutant variant was improved fourfold with respect to the wild-type enzyme. Based on the quantum molecular modeling and the increased activity of enzyme variants Ser105Ala and Ser105Gly, the authors proposed a reaction mechanism where the acidity of the α-hydrogen of the aldehyde was raised by coordination to the oxyanion hole of the enzyme, while the active site histidine (His) acted as a base to remove the α-hydrogen of the aldehyde. As the nucleophilic serine was removed, the basic nitrogen on the histidine was thought to get an increased ability to abstract the substrate’s α-proton. Then the enolate intermediate that is stabilized by hydrogen bond donors in the oxyanion hole was formed and reacted with a second molecule of aldehyde promoting the formation of the C-C bond (Scheme 13.2) (Branneby et al., 2004). Recently, Gotor and coworkers evaluated the promiscuous activity of commercially available protease from Bacillus licheniformis immobilized as cross-linked enzyme aggregates (Alcalase-CLEA) in the aldol addition between aromatic aldehydes and aliphatic ketones. Serious control reactions, including the use of bovine serum albumin in place of the enzyme,
the denaturation of Alcalase-CLEA for 1 h at 150°C, or with urea for 72 h at 150°C, and treatment of Alcalase-CLEA with phenylmethanesulfonyl fluoride (PMSF, a commonly used serine protease inhibitor) for 16 h at 30°C, were designed to confirm that the tertiary structure of the enzyme is required for the promiscuous aldol reaction (López-Iglesias et al., 2011). Aldol reactions catalyzed by promiscuous hydrolase have been well studied so far and have become one of the most successful promiscuous activities of hydrolase. In the early reports of Aldol reactions, no or very low enantioselectivity was observed. In the CAL-B-catalyzed aldol reaction of hexanal, the authors explained the possible reason for the low enantioselectivity to be the racemation of products (Branneby et al., 2004).
