title: Production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine by entrapped ACV-synthetase from Streptomyces clavuligerus.
abstract: delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV)-synthetase from Streptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45-65% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g-1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.
authors.affiliation: Department of Microbiology, University of Alberta, Edmonton, Canada.
authors.forename: T A
authors.initials: TA
authors.lastname: Kadima
authors.forename: S E
authors.initials: SE
authors.lastname: Jensen
authors.forename: M A
authors.initials: MA
authors.lastname: Pickard
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000187:drug effects / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007218:Industrial Microbiology; D008025:Ligases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D004800:Enzymes, Immobilized; D009842:Oligopeptides; C011638:5-(2-aminoadipyl)cysteinylvaline; D008025:Ligases; D003545:Cysteine