Immobilization of a non-heme chloroperoxidase from Serratia marcescens by entrapment into Ca-alginate beads and in semi-permeable protein membranes

Abstract: Chloroperoxidase (CPO) (EC 1.11.1.10) from Serratia marcescens W 250 was immobilized in Ca–alginate beads and semi-permeable membranes. The thermal stability, stability under storage and operational stability of the immobilized enzyme under determining halogenating and phosphatase activity have been reported. The addition of [Fe(CN6)]3- included into beads was found to increase the activity of the immobilized enzyme. Polyethylenimine(PEI)- and glutaraldehyde (GA) - coated beads showed good operational stability but the activity of the enzyme especially in the case of PEI greatly reduced. GA-coated beads with ferricyanide possess good stability under operational conditions and could be stored in the reaction buffer at 40C for at least 5 weeks. The most applicable variant of semi-permeable membranes was made of gelatin as a protein carrier and treated with 0.5 % GA for 2 min. The best variants of immobilized CPO preparations for determining halogenating and phosphatase activity were proved in the fluidized-bed column bioreactor. The yield of products was 91 % for GA-coated gelatin membranes with monochlorodimedone (MCD) and phenol red as substrates at the flow rate through the reactor of 27 ml/hr and they lost only about 20% of activity during 10 days. For Ca-alginate beads the yield of tetrabromphenol and p-nitrophenol (pNP) was 63 % and 56 % correspondingly at 32 ml/ hour. For pNP the yield stays stable at such a level during 10 days of the reactor work.