EFFICIENT DOWNSTREAM PROCESSING OF MALTODEXTRIN PHOSPHORYLASE FROM ESCHERICHIA-COLI AND STABILIZATION OF THE ENZYME BY IMMOBILIZATION ONTO HYDROXYAPATITE

Downstream processing by biospecific chromatography of maltodextrin ph osphorylase from Escherichia coli, overexpressed in E. coli, was subst antially improved by a novel approach using ceramic hydroxyapatite. Wi ld-type and a less active mutant enzyme were purified from crude bacte rial cell extracts in one efficient separation step that yielded phosp horylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxy apatite column at a phosphate concentration of 0.4 M whereas calcium i ons failed to displace the enzymes. The dynamic capacity for phosphory lase binding in the presence of bulk proteins was approximately 3 mg e nzyme m1(-1) matrix. The interaction of E. coli phosphorylase with hyd roxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational st ability of phosphorylase during conversion of polysaccharide to glucos e l-phosphate. (C) 1997 Elsevier Science B.V..