Enzyme surface glycosylation in the solid phase: Improved activity and selectivity of Candida antarctica lipase B

Tailor-made oligosaccharides and polymers were investigated for a specific surface glycosylation of Candida antarctica lipase (fraction B) (CAL-B) already immobilized on octyl-Sepharose by interfacial activation. The chemical modification was performed in the N-terminal amino acid enzyme residue by using low oxidized aldehyde-dextran polymers through a reductive amination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. The formed immobilized glycosylated lipase biocatalysts were more stable, active, and selective toward different substrates than unmodified CAL-B. These immobilized conjugates were compared with a genetically glycosylated version of CAL-B expressed in Pichia pastoris immobilized in the same way. Enzyme thermostability was improved after chemical modification with Dextran-1500 and also by the genetic glycosylation, retaining 90-96% activity after 24h at 55°C. The catalytic activity of CAL-B was improved by the incorporation of dextran polymers (M_w=1500 or 6000) more than twofold in the hydrolysis of p-nitrophenylbutyrate and more than threefold in the hydrolysis of methyl mandelate at pH7. However, the activity of the genetically glycosylated CAL-B was threefold lower in the hydrolysis of both substrates. The enantioselectivity of CAL-B increased for all formed bioconjugates, with the Dextran-1500-CAL-B conjugate being the most selective in the hydrolysis of racemic methyl mandelate (up to 88.1% ee at pH5). This glycosylated CAL-B also demonstrated the highest synthetic activity in the transesterification of methyl butyrate with glycerol, with 80% yield of monoglyceryl ester at 100% conversion compared to 57% yield obtained with unmodified CAL-B or other polymer-lipase conjugates.