EFFICIENT DOWNSTREAM PROCESSING OF MALTODEXTRIN PHOSPHORYLASE FROM ESCHERICHIA-COLI AND STABILIZATION OF THE ENZYME BY IMMOBILIZATION ONTO HYDROXYAPATITE
C. Eis et al., EFFICIENT DOWNSTREAM PROCESSING OF MALTODEXTRIN PHOSPHORYLASE FROM ESCHERICHIA-COLI AND STABILIZATION OF THE ENZYME BY IMMOBILIZATION ONTO HYDROXYAPATITE, Journal of biotechnology, 58(3), 1997, pp. 157-166
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..