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

Penicillin G acylase (PGA) from Escherichia coil was cross-linked with mann an 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 m annans with different degrees of oxidation and weight-average molecular mas s (M-W). The pH- and thermostability of all preparations of glycosylated en zyme were improved remarkably, whereby the influence of the character of th e linked mannan dialdehyde, its content, as well as the molecular mass of p repared glycoconjugates, on the stability of PGA, was evaluated. PGA glycos ylated 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, s howed the highest stability. The half-life of this PGA preparation increase d significantly: 13-fold at pH 3, 7-fold at pH 10, and 3.5-fold at pH 8 (al l at 37 degreesC), compared with the native enzyme. At higher temperatures (50 degreesC) even more significant stabilization was evident, a 16-fold in crease in half-life, from 18 min to 289 min, at pH 8, being measured.