Comparative characterization of deletion derivatives of the modular xylanase XynA of Thermotoga maritima.

The modular Xylanase XynA from Thermotoga maritima consists of five domains (A1-A2-B-C1-C2). Two similar N-terminal domains (A1-A2-) are family 22 carbohydrate-binding modules (CBMs), followed by the catalytic domain (-B-) belonging to glycoside hydrolase family 10, and the C-terminal domains (-C1-C2), which are members of family 9 of CBMs. The gradual deletion of the non-catalytic domains resulted in deletion derivatives (XynADeltaC; XynADeltaA1C and XynADeltaNC) with increased maximum activities (V (max)) at 75 degrees C, pH 6.2. Furthermore, these deletions led to a shift of the optimal NaCl concentration for xylan hydrolysis from 0.25 (XynA) to 0.5 M (XynADeltaNC). In the presence of the family 22 CBMs, the catalytic domain retained more activity in the acidic range of the pH spectrum than without these domains. In addition to the deletion derivatives of XynA, the N-terminal domains A1 and A2 were produced recombinantly, purified, and investigated in binding studies. For soluble xylan preparations, linear beta-1,4-glucans and mixed-linkage beta-1,3-1,4-glucans, only the A2 domain mediated binding, not the A1 domain, in accordance with previous observations. The XynA deletion enzymes lacking the C domains displayed low affinity also to hydroxyethylcellulose and carboxymethylcellulose. With insoluble oat spelt xylan and birchwood xylan as the binding substrates, the highest affinity was observed with XynADeltaC and the lowest affinity with XynADeltaNC. Although the domain A1 did not bind to soluble xylan preparations, the insoluble oat spelt xylan-binding data suggest that this domain does play a role in substrate binding in that it improves the binding to insoluble xylans.