Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan

Penicillin G acylase (PGA) from Escherichia coli was cross-linked with mannan dialdehydes. Conjugates were prepared with molecular masses varying from 140 to 580 kDa and containing from 18 to 50% (w/w) saccharides, the values depending on the reaction conditions (mannan/enzyme ratio), and by using mannans with different degrees of oxidation and weight-average molecular mass (M-W). The pH- and thermostability of all preparations of glycosylated enzyme were improved remarkably, whereby the influence of the character of the linked mannan dialdehyde, its content, as well as the molecular mass of prepared glycoconjugates, on the stability of PGA, was evaluated. PGA glycosylated with the most oxidized mannan up to an Mw of 490 kDa, containing 41% (w/w) saccharides, and retaining 90% of its original catalytic activity, showed the highest stability. The half-life of this PGA preparation increased significantly: 13-fold at pH 3, 7-fold at pH 10, and 3.5-fold at pH 8 (all at 37 degreesC), compared with the native enzyme. At higher temperatures (50 degreesC) even more significant stabilization was evident, a 16-fold increase in half-life, from 18 min to 289 min, at pH 8, being measured.