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.
Highlights
A fusion protein is a molecule consisting of two or more protein domains incorporated into one single complex
Primers were synthetized by Microsynth AG; NADH and NAD+ were purchased from Apollo Scientific Ltd., while immobilization supports were kindly provided by Resindion S.r.l and Purolite Ltd
In silico analysis have been performed to predict the assembly of the fusion protein, when a short peptide linker of GSGGGGSAS is integrated between the two protein domains
Summary
A fusion protein is a molecule consisting of two or more protein domains incorporated into one single complex. Artificial fusion proteins may be designed to achieve improved properties or new functionality (Yu et al, 2015) For this purpose, the genetic combination of two proteins to generate a bifunctional enzyme complex has evolved over the years (Lindbladh et al, 1992). The creation of one single protein presenting the biocatalytic activity of two distinct enzymes can dramatically simplify the phases of expression and purification with a significant impact on time, materials and costs spent for the manipulation. This feature translates into sustainable processes that are extremely attractive in line with the growing demand of environmental protection policies
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