Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and L-N-carbamoylase from Arthrobacter aurescens for the production of optically pure L-amino acids

The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C: 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharuse-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements. In comparison to the free enzymes, temperature and pH-optima were increased by 10 degreesC and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degreesC (60 degreesC). In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor. (C) 2001 Elsevier Science Inc. Atl rights reserved.