Studies On Immobilization Of Enzyme And Cells

Enzyme immobilization has attracted a wild range of interest from fundamental academic research to many different industrial applications. The immobilization can offer several advantages, including repeated use, ease of separation of reaction products from the biocatalyst, improvement of enzyme stability, etc. For this reason, the immobilization and stabilization of the hydantionase were major importance for the development of a progress for the production of unnatural D-amino acids from D,L-monosubstituted hydantoins.Optically active D-amino acids are widely used as intermediates for synthesis of semisynthetic antibiotics, peptide hormones, pyrethroids and pesticides. D-amino acids are usually produced by enzymatic or chemo-enzymatic method. The DL-5'-monosubstitued hydantoin is enantioselectively hydrolyzed to the corresponding N-carbamoyl-D-amino acid by D-hydantoinase, and this intermediate is further converted to the free D-amino acid by D-Carbamoylase or chemical decarbamoylation. Because chemical decarbamoylation has several problems such as low yield and large disposal of waste, much attention has been paid to N-carbamoylase for development of an economic process.The immobilization of engineered cells producing D-hydantoinase was performed to explore the bioconversion of D-amino acid with DL-p-Hydroxyphenylhydantoin(pHPH) as the substrate. The optimal condition of cell immobilization was that 5 grams of 2.5% alginate sodium were mixed with cells corresponding to 30mL bacterial culture, and then the suspended solution was dropped into 5% CaCl₂ solution to form beads which were harded at 4°C for 5 hours. The enzyme activity of immobilization cells obtained as the above manipulation could reach 219% as compared with the equal amount of free cells. Its optimal pH is near pH 9.0 as the same as free cells, but more stable at the alkaline side. The thermal stability is obviously...