Biotechnology Techniques Vol 3 No 2 135-138 (1989) Received December 20 STUDY OF A BACTERIAL HYDANTOINASE (DIHYDROPYRIMIDINASE) ACTIVITY USING ALGINATE BEADS AND A CELL RECYCLE SYSTEM P. Chevalier, D. Roy*, and A. Morin Agriculture Canada, St-Hyacinthe Food Research Center, St-Hyacinthe, Qu6bec, Canada J2S 8E3 SUMMARY This report describes the production of N-carbamyl- b -alanine, by resting cells of Pseudomonas p_utida, in two types of continuous fermentation processes: alginate- immobilized cells in a continuous stirred tank reactor and a free cell recycle system using microporous membranes. At pH 9,0 both systems resulted in a more efficient substrate conversion and a higher yield than at pH 8,0 although they had a similar performance. INTRODUCTION The enzyme dihydropyrimidinase (EC 3.5.2.2) has a wide substrate specificity on 5- substituted hydantoins (Yamada et al., 1978) and dihydrouracil (Morin et al., 1986a). This finding suggested the development of enzymatic methods for the production of optically active N-carbamyl derivatives of D-amino acids. However, dihydropyrimidinase (hydantoinase) is an intracellular enzyme which is unstable upon purification and when used in a free state (Morin et al., 1986b). Previous experiments showed that hydantoinase from Pseudomonas putida was more stable with non-growing immobilized whole cells used in packed bed columns (alginate beads) and in hollow fiber cartridges (Chevalier et al., 1989). In the present study we investigated alginate-immobilized cells of _P. putida in a continuous stirred tank reactor (CSTR) and free organisms in a cell recycle system using microporous membranes in a crossflow mode. MATERIAL AND METHODS Microbiology_. Biomass of P. ~ DSM 84 was obtained in medium M-1 (g.L-1): technical yeast extract, 5,0;-glycero b 5,0 and KgHPO 4, 2,0; pH was adjusted to 7,0 with KH2PO 4. After 72 h of growth (30~ at 150 rpm in 1L shaking flasks, containing 250 mL of medium, cells were harvested by centrifugation (4400 x g). 135