Abstract
Arthrobacter crystallopoietes produces a D-specific hydantoinase and
D-N-carbamoylase useful for the industrial production of
enantiomerically pure D-amino acids. The D-hydantoinase was purified by
conventional chromatographic methods. The enzyme was digested by
trypsine and some of the oligopeptides sequenced. The amino acid
sequence information was used to isolate a DNA fragment of the
corresponding gene. By several steps of inverse PCR a region of 6011 bp
was obtained from the A. crystallopoietes genome surrounding the
hydantoinase gene fragment. DNA sequence analysis revealed the presence
of a D-hydantoinase, the D-N-carbamoylase and a putative
L-N-carbamoylase on the 6011 bp fragment. In addition, two incomplete
open reading frames (ORFs) showed similarity to LacI type
transcriptional regulators and transmembrane proteins responsible for
the uptake of nucleosides and allantoin, respectively. The
D-hydantoinase gene and the D-N-carbamoylase gene were expressed in
Escherichia coli and the enzyme activity was determined. From the
putative L-N-carbamoylase gene, expressed in the same way in E. coli,
only insoluble protein was obtained and no carbamoylase activity was
detected.
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