Cloning, nucleotide sequence and expression of a new L-N-carbamoylase genefrom Arthrobacter aurescens DSM3747 in E-coli

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthroba cter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence w as determined. After expression of the gene in E, coli the enzyme was purif ied to homogeneity and characterized. The enzyme was shown to be strictly L -specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N-alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Car bamoyl derivatives of beta-alanine and charged aliphatic amino acids were P lot accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 dif fers from all known enzymes with respect to its substrate specificity altho ugh amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44.000 Da, and h as an isoelectric point of 4.3. The optima of temperature and pH were deter mined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzym e was completely stable for several days. (C) 1999 Elsevier Science B.V. Al l rights reserved.