Immobilization of D-Hydantoinase on Eupergit C and Eupergit C 250L.
Sarante, N ; Oestreicher, E. G.
Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Ilha do Fundão , Rio de Janeiro 21945-970.
D-hydantoinase from Vigna angularis was immobilized by covalent linkage to the oxirane polimer Eupergit. To type of this support were used: Eupergit C and Eupergit C 250. This procedure was undertaken in order to obtain an enzyme preparation stable and with high activity for the preparation of non-proteinogenic amino acids. The target product were: D-phenylglycine (one of the component of the antibiotic ampicillin); D-p-hydroxy-phenylglycine (component of the antibiotic amoxicillin).These target product were obtained by enantioselective hydrolysis of the respective racemic hydantoins (phenylhydantoin and p-hydrophenyl hydantoin) which in turn were synthesized from benzaldehyde (or p-hydeoxybenzaldehyde with KCN and (NH4)2+CO3.Enzymatic activity was measured at pH 9.0 (buffer: 100 mM H3BO3/KCl), 30 oC with 67 mM of substrate and an enzyme concentration required to maintain the reaction under initial velocity conditions, for 5 min at least. The product formed was detected with a colorimetric assay using p-dimethyl aminoabenzaldehyde as reagent. Immobilization in both epoxy-activated supports was performed with a procedure involving to steps. Initially to 500mg of support (Eupergit C or Eupergit C-250L, suspended in 5 mL of 100mM H3BO3/KCL, pH 9.0 buffer were incubated at 30oC under orbital shaking with 200 mg of D-hydantoinase from Vigna angularis during 48 h.. The progress of immobilizations was followed by measuring the enzyme activity and protein concentration in aliquots of the supernatant fluid. The yield of this singlepoint immobilization was of 22.8% in both support. The immobilized enzyme was then submitted to a multipoint attachment to the support by incubation at 20oC in KHCO3/K2CO3 buffer, pH 11.0. The yield of this step was 63% approximately, for both supports. The enzyme immobilized is stable when stocked at 5oC during all the time tested but was inactivated when stocked at -5oC during 24 h.
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