The binding pattern of two carbohydrate-binding modules of laminarinase Lam16A from Thermotoga neapolitana: differences in beta-glucan binding withinfamily CBM4

Carbohydrate-binding modules (CBMs) are often part of the complex hydrolyti c extracellular enzymes from bacteria and may modulate their catalytic acti vity. The thermostable catalytic domain of laminarinase Lam16A from Thermot oga neapolitana (glycosyl hydrolase family 16) is flanked lay two CBMs, 148 and 161 aa long. They share a sequence identity of 30%, are homologous to family CBM4 and are thus called CBM4-1 and CBM4-2 respectively. Recombinant Lam16A proteins deleted for one or both binding modules and the isolated m odule CBM4-1 were characterized. Proteins containing the N-terminal module CBM4-1 bound to the soluble polysaccharides laminarin (1,3-beta -glucan) an d barley 1,3/1,4-beta -glucan, and proteins containing the C-terminal modul e CBM4-2 bound additionally to curdlan (1,3-beta -glucan) and pustulan (1,6 -beta -glucan), and to insoluble yeast cell wall P-glucan. The activity of the catalytic domain on soluble 1,3-beta -glucans was stimulated by the pre sence of CBM4-1, whereas the presence of CBM4-2 enhanced the Lam16A activit y towards gelatinized and insoluble or mixed-linkage 1,3-beta -glucan. Ther mostability of the catalytic domain was not affected by the truncations. Me mbers of family CBM4 can be divided into four subfamilies, members of which show different polysaccharide-binding specificities corresponding to the c atalytic specificities of the associated hydrolytic domains.