Fusion of Glutamate Dehydrogenase and Formate Dehydrogenase Yields a Bifunctional Efficient Biocatalyst for the Continuous Removal of Ammonia

Abstract

A novel fusion protein has been rationally designed, combining the hexameric glutamate dehydrogenase from Clostridium symbiosum with the dimeric formate dehydrogenase from Candida boidinii. The former enzyme consumes ammonia for the reductive amination of α-ketoglutarate using NADH, while the latter biocatalyst regenerates continuously the cofactor. This enzymes fusion opens new perspectives for the detection and the removal of ammonia. The bifunctional biocatalyst has been successfully created, expressed, and then characterized. The two fused protein domains retained identical properties and catalytic activity of the individual enzymes. Additionally, the immobilization on a methacrylate resin optimized the assembly providing a reusable and stable biocatalyst. This is an example of immobilization of a fusion protein, so that efficiency and sustainability of the process are enhanced. The immobilized biocatalyst could be recycled 10 times retaining still half of the initial activity. Such preparation outperforms the co-immobilized wild-type enzymes in the conversion of 300 mM of ammonia, which could be carried out also in continuous mode.

Fusion of Formate Dehydrogenase and Alanine Dehydrogenase as an Amino Donor Regenerating System Coupled to Transaminases

“…Indeed, the presence of glycine and serine as small polar amino acids has been shown to provide good flexibility and optimal stability in water [23] . A (6x)His‐tag was also fused to the FDH domain for purification purposes as in a previous fusion protein including the CbFDH [19] …”

“…A similar behavior was found in other fusion proteins. The GluDH domain in the GluDH‐FDH protein presented a K M 2‐fold higher than the parental enzymes [19] . Similarly, the PheDH‐FDH‐His fusion protein had 2‐fold higher K M values for the substrates phenylpyruvate and formate than the WT enzymes [31] .…”

“…A linker of Gly‐Ser‐Gly‐Gly‐Gly‐Gly‐Ser‐Ala‐Ser was included between the two domains. Four restriction sites were added as reported for a previous fusion protein construction [19] . The plasmid of the CbFDH (UniProt ID: O13437) was used as template for PCR amplification (vector fragments).…”

“…Despite the advantages offered by multi‐enzyme systems, the production and optimization of each individual enzyme is costly and time‐consuming. Fusion proteins can address these difficulties by combining enzymes that are catalytically compatible into one single multifunctional enzyme, [16,17] and the genetic fusion can result into improved catalytic efficiency due to the closer proximity of the active sites [18,19] . Additionally, gene expression, folding, and enzyme stability can be potentially enhanced, but the design of the fusion enzyme must be carefully rationalized [16] …”

More efficient enzymatic cascade reactions by spatially confining enzymes via the SpyTag/SpyCatcher technology

“…It has been pointed out that the spatial confinement of different biocatalysts operating in parallel or in sequence can drastically reduce diffusion distances of the reagents and thereby accelerate the rate of biocatalytic cascade reactions [2]. Next to co-immobilization of different enzymes [3] or confinement in vesicles [4] also genetically fused enzymes [2,5,6] have been investigated. From these studies it can be concluded that generally, spatial proximity represents an advantage for cascade catalysis as diffusion distances of the individual reagents are reduced.…”