Purification of D-hydantoinase from adzuki bean and its immobilization for N-carbamoyl-D-phenylglycine production

Hydantoinase could be extracted from adzuki bean by a simple separation process. The molecular weight of the partially purified hydantoinase determined by MALDI-TOF mass spectrometry was 52.5 kDa. The enzyme was determined to be D-specific and preferred the substrate D-phenylhydantoin (PH) rather than D-p-hydroxy-phenylhydantoin (pHPH). Its specific activity towards PH was about sixfold of that towards pHPH. The enzyme retained 76% of its activity after incubation at 40 degreesC for 6 days. Its immobilization was easily achieved by mixing the enzyme solution with fine polyglutaraldehyde (PGL) particles (< 10 mum). In order to enlarge the size of the immobilized enzymes for easy recovery, the fine immobilized enzyme particles were then entrapped in the calcium alginate bead and hardened with polyethyleneimine (PEI). The immobilized D-hydantoinase could transform 1% (w/v) PH into N-carbamoyl-D-phenylglycine (D-CPG) with > 95% conversion and very good stability that no appreciable activity loss was observed after five repeated batch reactions at 40 degreesC. (C) 2001 Elsevier Science B.V All rights reserved.