title: Construction and evaluation of a novel bifunctional N-carbamylase-D-hydantoinase fusion enzyme.
abstract: 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 bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the D-hydantoinase. Firstly, maltose-binding protein (MBP) gene of E. coli was fused with D-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of native D-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics of D-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of N-carbamylase (304 amino acids [aa]) from Agrobacterim radiobacter NRRL B11291 and D-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to the D-amino acids. A related D-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene 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 fusion 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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-dong, Yusung-gu, Taejon 305-701, Korea.
authors.forename: G J
authors.initials: GJ
authors.lastname: Kim
authors.forename: D E
authors.initials: DE
authors.lastname: Lee
authors.forename: H S
authors.initials: HS
authors.lastname: Kim
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D000581:Amidohydrolases; C409187:N-carbamylase-D-hydrantoinase; C020047:dihydropyrimidinase