Expanding the bioanalytical application of β‐hydroxybutyrate binding proteins through characterization of their metabolite interactions and site‐directed mutagenesis

β-hydroxybutyrate binding proteins (BHBBPs) are a newly identified group of periplasmic solute-binding proteins (SBPs) that interact with β-hydroxybutyrate (BHB), a key physiological metabolite. In this study, we systematically characterized the interaction properties of both previously reported and newly identified BHBBPs, including "NovoS" and "EDC10" from Gram-negative bacteria. Following recombinant production, we assessed the specificity and affinity of these proteins against a library of 23 different metabolites using a label-free derivative of differential scanning fluorimetry (nanoDSF). Positive interactions were further evaluated for their binding affinity via tryptophan fluorescence spectroscopy, which confirmed D/L-BHB as the preferred ligand for all proteins, with slight enantioselectivity. BHBBPs also exhibited binding to other compounds such as acetoacetate, D/L-α-hydroxybutyrate, L-lactate, and pyruvate, albeit with reduced affinity. These findings expand the classification of BHBBPs, suggesting that similar proteins and associated transporters may be widespread in prokaryotes involved in the carbon cycle of polyhydroxybutyrate. Guided by the crystal structure of the homologous BMA2936 protein, we introduced targeted point mutations in conserved polar residues of the BHBBPs EDC24 and NovoS. It was determined through this experimental pipeline that their affinity towards BHB was reduced by a factor between 25 and 750, shifting their binding constants towards the millimolar range. Collectively, the affinities of both wild-type and mutant proteins span 4 orders of magnitude, from nanomolar to millimolar recognition of BHB. Leveraging the versatility of SBP-based biosensing, these receptors and their wide affinity range could facilitate the development of effective bioanalytical tools for BHB detection in diverse physiological environments.