From Identification to Enzymatic Activity Profiling: Comprehensive Characterization of BloCAβ, a Probiotic β-Carbonic...

The microbiota comprises communities of microorganisms that establish complex relationships with the host and play key roles in physiology, metabolism, and immunity. Increasing evidence highlights microbiota alterations in several diseases, particularly those driven by oxidative stress-induced inflammation. Thus, identifying probiotic enzymes implicated in essential metabolic pathways is highly relevant to the development of future strategies targeting dysbiosis and related diseases. Herein, we report the first biochemical characterization of β-class carbonic anhydrase (CA) from Bifidobacterium longum (BloCAβ) by inhibition and activation profiles. The enzyme was recombinantly expressed, purified, and kinetically characterized, displaying a catalytic efficiency within the same order of magnitude as, although lower than, that of human (h)CA I. The functional profile of this novel CA was explored using a panel of commercial sulfonamides, sulfamides, sulfamates, and inorganic/organic anions as inhibitors. Furthermore, several biogenic amines and amino acids were assessed as potential activators of BloCAβ, and their activation profiles were compared with those of hCAs I and II. The cellular effects of selected BloCAβ activators was further evaluated in human macrophages under basal and proinflammatory conditions, while the safety profile was assessed in human intestinal epithelial cells (HIECs). Among them, serotonin (38) was found to significantly enhance cell metabolic activity in both conditions and preserve the metabolic activity of HIECs, further supporting its biocompatibility. Overall, these results provide the first biochemical and functional characterization of BloCAβ and establish a biochemical basis for future studies investigating the physiological role of probiotic CAs in bacterial models.