Summary: The obligately methylotrophic bacterium Methylophilus methylotrophus readily uses formamide as a source of nitrogen for growth. Physiological investigations using batch, fed-batch and continuous cultures indicated that the organism contains a discrete formamidase (formamide amidohydrolase; EC 3.5.1.49), in addition to the previously characterized aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) which is specific to short-chain aliphatic amides such as acetamide, propionamide and acrylamide. Formamidase synthesis was induced by formamide and acetamide, and repressed by ammonia. The enzyme was purified using anion-exchange and gel-filtration FPLC and shown to exhibit a narrow substrate specificity (high activity with formamide, little activity with short-chain aliphatic amides, no activity with urea). SDS-PAGE and gel-filtration FPLC showed that the enzyme comprises a single type of subunit and probably exists as a homodimer. A 3·2 kbp Pstl restriction fragment of chromosomal DNA from M. methylotrophus was cloned in pUC19 and expressed in Escherichia coli JM109. The purified gene product exhibited essentially identical properties to those of the M. methylotrophus formamidase in terms of its chromatographic behaviour, native Mr (123 000), subunit Mr (51 000), Kcat (58 cf. 64 s−1), Km (1·6 cf. 2·1 mM), substrate specificity and N-terminal amino acid sequence (MKTIV-).
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