The specificity of family 4 carbohydrate-binding modules (CBMs) of BpGH16MLG influences the binding and utilization of β-glucans in a human gut bacterium, Blautia producta

Several thousand bacterial communities reside in the human gut environment and have acquired numerous genes during co-evolution for trapping dietary nutrients. Many of Gram-positive bacteria encode extracellular cell wall membrane-bound endolytic enzymes with non-catalytic carbohydrate-binding modules (CBMs) that recognize cognate glycan(s). Much remains unknown about how these organisms recognize nutrients in the gut environment. Blautia producta JCM 1471^T expresses a cell membrane-tethered endo-acting enzyme (BpGH16_MLG-digesting barley-β-glucan, lichenan, and laminarin) with four carbohydrate-binding modules (CBM4_1, CBM4_2, CBM4_3, and CBM4_4). Each CBM4 and its dual combinations were cloned, characterized, and assessed with three distinct β-glucans (barley-β-glucan, lichenan, and laminarin). It was observed that each CBM4 recognizes barley-β-glucan and lichenan with approximately equal affinity. Dual combination, CBM4_1-CBM4_2 exhibited a substantial predilection for barley-β-glucan, whereas CBM4_3-CBM4_4 had an affinity for barley-β-glucan and lichenan. None of the dual combinations showed an affinity for laminarin. Intriguingly, a mutant (E384A-catalytic base) version of BpGH16_MLG with all CBM4 domains showed high binding to barley-β-glucans and lichenan, but moderate binding to laminarin. This understanding provides insights into how B. producta recognizes and likely traps specific nutrient in the gut environment. Furthermore, the study may have implications for the development of innovative biocatalysis.