Properties of four C-terminal carbohydrate-binding modules (CBM4) of laminarinase Lic16A of Clostridium thermocellum

At the C-terminus of multimodular laminarinase Lic16A from Clostridium thermocellum, four carbohydrate-binding modules (CBM) of family 4 were found. Isolated CBM4_1, CBM4_2, CBM4_3, and CBM4_4 modules and the CBM4_(1-4) tandem were obtained. None of the recombinant proteins had the affinity to soluble ??-1,3-1,4-glucans, laminarin and lichenan, the main specific Lic16A substrates. All modules, except CBM4_4, had the ability to bind bacterial crystalline cellulose, which is atypical of family-4 CBMs. All CBMs 4 of Lic16A had an affinity to xylan, chitin, yeast cell wall ??-glucan, and avicel, while CBM4_3 and CBM4_4 also had an affinity to chitosan. The CBM4_(1-4) tandem had the highest affinity to the ??-glucan, avicel, and pustulan of the yeast cell wall. The CBM4_(1-4) binding constants for these substrates were approximately 100-fold higher than those of its individual modules, which suggests synergy in the process of absorbing these polysaccharides. This finding helps to explain the evolutionary process of CBM multiplication.