title: Stability enhancement of Escherichia coli penicillin G acylase by glycosylation with yeast mannan.
abstract: 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 macro(w)). The pH- and thermo-stability 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 M(w) 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 degrees C), compared with the native enzyme. At higher temperatures (50 degrees C) even more significant stabilization was evident, a 16-fold increase in half-life, from 18 min to 289 min, at pH 8, being measured.
authors.affiliation: Institute of Chemistry SAS, Dúbravská cesta 9, SK-842 38 Bratislava, Slovak Republic. chemjmas@savba.sk
authors.forename: J
authors.initials: J
authors.lastname: Masárová
authors.forename: D
authors.initials: D
authors.lastname: Mislovicová
authors.forename: P
authors.initials: P
authors.lastname: Gemeiner
authors.forename: E
authors.initials: E
authors.lastname: Michalková
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008351:Mannans / Q000378:metabolism*; D010399:Penicillin Amidase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D008351:Mannans; D010399:Penicillin Amidase