Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747

The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Art hrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hy uC2) were cloned in Escherichia coli and the nucleotide sequences determine d. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase g enes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylas es, showed a high degree of nucleotide and amino acid sequence identity (96 -98%). The hyuH and hyuC genes were expressed in E. Loll under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original host s. For purification the hyuH2 gene was expressed as a maltose-binding prote in (MalE) and as an intein-chitin binding domain (CBD) fusion in E. coli. T he expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optim al temperature, substrate and ste reospecificity, the purified fusion enzym e exhibited properties similar to those of the wild-type enzyme.