Hydantoin-hydrolyzing enzymes of Agrobacterium tumefaciens isolates such as strain RU-OR are used as biocatalysts in the commercial production Of D-hydroxyphenylglycine via hydrolysis Of D, L-p-hydroxyphenylhydantoin. Hydantoin-hydrolyzing enzyme activity in RU-OR cells is tightly regulated by nitrogen catabolite repression and is induced when hydantoin or a hydantoin-analogue is present in the growth medium. Previous studies have selected mutant strains which are inducer-independent and no longer Subject to nitrogen catabolite expression. However, these mutants did not exhibit significantly higher levels of enzyme activity compared to the wild-type strain. In this Study, we have focused on enhancing the levels of hydantoinase and N-carbamoylase activity in wild-type RU-OR cells by manipulating the growth medium or over-expressing the global nitrogen regulatory factors, NtrBC. We also show that this strain encodes two distinct D-selective N-carbamoylases. One enzyme is virtually identical to the other Agrobacterium N-carbamoylases while the second represents a new class Of D-N-carbamoylases with potentially novel biocatalytic properties. (C) 2008 Elsevier Inc. All rights reserved.