Construction and Evaluation of a Novel Bifunctional N -Carbamylase– d -Hydantoinase Fusion Enzyme

Abstract

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.In the field of molecular biology and biotechnology, enzymes possessing two or more combined activities, along with appropriate stability, have found wide application (25). Although the natural diversity of the enzymes provides some candidates that have evolved to possess bifunctional activity, most fusion enzymes have resulted from the in vitro fusion of individual enzymes based on evolutionary traits and well-defined structure (1, 25). Artificial fusion enzymes, simply generated by either end-to-end fusion or by tethering of whole genes encoding intact functional proteins with a linker, have been reported to show noticeable performance in a concerted fashion (6, 21).Nonnatural D-amino acids are widely used in the pharmaceutical field, with applications such as antimicrobial and antiviral agents, artificial sweeteners, pesticides, and pyrethroids (26,28,30). Due to the great commercial demand for various D-amino acids, enzymatic and chemoenzymatic routes have been developed (29). Of these, a fully enzymatic process using two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity (8). In this process, hydantoin derivative is hydrolyzed by D-hydantoinase, and the resulting N-carbamyl-D-amino acid is further converted to the corresponding D-amino acid by N-carbamylase. Therefore, functional fusion of two enzymes was expected to have several advantages over individual enzymes with respect to reaction kinetics and enzyme production, as well as novel properties and reactivity. As practical cases of the multistep sequential reaction, the performance of the hybrid or fusion enzymes sometimes was better than that achieved by successive action of individual enzymes, expanding the potential use of natural enzymes (6,21,25).Previously, we cloned and expressed two hydantoinase genes from Bacillus stearothermophilus SD1 (17) and Bacillus thermocatenulatus GH2 (15). We also identified some conserved domains possessing essential amino acid residues by comparative analyses of functionally related enzymes (16). These results provided some evidence that supports the presence of a cyclic amidohydrolase family including hydantoinase, dihydropyrimidinase, allantoinase, and dihydroorotase. Further study of the deletion mutants derived from two D-hydantoinases suggested that the N terminus of D-hydantoinase is not essential for maintaining the enzyme structure and is dispensable for enzyme activity (15,17). From these observations, we made a hypothesi...

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

“…This is the same as for the BTS1-ERG20 fusion gene. Such a proximity effect and substrate channeling for fusion enzymes have been reported to increase the overall catalytic activity of the reaction in vitro (11,15,17,33). We demonstrated that the use of bifunctional fusion genes was also highly effective for metabolic engineering.…”

Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway

“…in the presence of plasmid pMCS69 was therefore likely to be due to the inefficient fusion of PhaA to PhaB. This was not unexpected as both PhaA and PhaB enzymes function catalytically as homo-tetramers (Davis et al, 1987;Ploux et al, 1988) so fusions of these could reduce the activity by interfering with quaternary structures (Carlsson et al, 1996;Kim et al, 2000).…”

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris

“…It is generally recognized that closer spatial distance between the fused proteins affects their folding, function, and expression. Flexible linkers are typically incorporated in cases where the fused domains require a certain degree of movement or interaction (Kim et al, 2000;Blazyk and Lippard, 2004;Jiang et al, 2005;Wang et al, 2007;Lu and Feng, 2008). Most commonly used flexible linker sequences primarily consist of Gly and Ser residues ("GS" linker) (Huston et al, 1988;Kavoosi et al, 2007;Hölsch and Weuster-Botz, 2010).…”