Construction and evaluation of a novel bifunctional N-carbamylase-D-hydantoinase fusion enzyme

A fully enzymatic process employing two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity for the production of D-amino acids. To test the possibility of generating a b ifunctional fusion enzyme, we constructed a fusion protein via end-to-end f usion of a whole gene that encodes an intact protein at the N terminus of t he D-hydantoinase. Firstly, maltose-binding protein (MBP) gene of E. coli w as fused with D-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared wit h those of native D-hydantoinase. Gel filtration and kinetic analyses clear ly demonstrated that the typical characteristics of D-hydantoinase are main tained even in a fusion state. Based on this result, we constructed an arti ficial fusion enzyme composed of the whole length of N-carbamylase (304 ami no acids [aa]) from Agrobacterim radiobacter NRRL B11291 and D-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an e xpected molecular mass of 86 kDa and efficiently converted exogenous hydant oin derivatives to the D-amino acids. A related D-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase g ene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fus ion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.