Molecular Microbiology (1995) 15(3), 431-444 identificatSori of a novel ceilulose-binding domain the multidomain 120 kDa xylanase XyoA of the hyperthermophilic bacterium Thermotoga maritima Christoph Winterhalter,^ Peter Heinrich,^ Anton Candussio,^ Gunther Wich^ and Wolfgang Li'ebf'* ^ Lehrstuhl fur Mikrobiologie. Techniscbe Universitat Munchen. AroisstraBe 21. 0-80290 Munchen, Germany. • ^Gonsortium fur Eiektrochemische industrie GmbH, Munchen, Germany. Summary A segment of Ttiermotoga maritima strain I\/ISB8 chromosomal DNA was isolated which encodes an endo-1,4-(J-o-xy!anase, and the nucleolide sequence of the xylanase gene, designated xynA, was deter- mined. With a half-life of about 40 min at 9O'C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059-amino-acid (--120 kDa) modular enzyme composed of an W-termina! signal peptide and five domains, in the order A1-A2-B-C1-C2. By comparison witb other xylanases of family 10 of glycosyl hydro- lases, the central - 340-amino-acid part (domain B) of XynA represents the catalytic domain. Tbe N- terminal -150-amino-acid repeated domains (A1-A2) have no significant similarity to the C-terminal -.170- amino-acid repeated domains (C1-C2). Cellulose- binding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystal- line cellulose and that C2 alone can also promote cel- lulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulose- binding domain (CBD) and thus are CBDS of a novel type. Structurally related protein segments whicb are probably also CBDs were found in other multi- domain xylanolytic enzymes. Deletion of the W- terminal repeated domains or of all the non-catalytic domains resulted In substantially reduced tbermo- stability while a truncated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme. It is argued that tbe multi- domain organization of some enzymes may be one Received 9 June, 1994: revised 6 Octobei, 1994; accepted 7 October, 1994, *For correspondence, Tel. (89) 21052378; Fax (89) 21052360. of tbe strategies adopted by thermopbiles to protect their proteins against thermal denaturation. Introduction Xylan, in contrast io the other main structural polysac- charide of plant cell walls, i.e, cellulose, has a r'ela- tively complex structure based on a non-branched [1-1,4- glycosidically linked xylose backbone, Dependirig on tbe origin, the backbone slructur-e is substituted to varying degrees with acetyl, L-arabinofuranosyi, glucuronyl or 4- O-mefhylglucuronyl side-chain groups (see Coughlan and Haziewood, 1993), The two enzymes generally involved in main-chain breakdown by microorganisms are endo-1,4-p-D-xylanase (1,4-fi-D-xylan-xvlanohydro- lase, EC 3.2,1,8) and [i-xylosidase (|i-o-xyloside-xylo- hydrolase. EC 3.2,1.37), In this communication, we report for the fir'st time the cioning of a xylanase gene from a hyperthermophilic organism, Thermotoga niari- tirna, and describe the nucleotide sequence-derived pri- mary siructure and some biochemical properties of the thermostable recombinant enzyriie, A variety of cellulolytic and hemicellulolytic enzymes have mullidomain structures wilh one (in some cases two) catalytic dornain(s) and one or more non-catalytic domains (Gilkes et ai, 1991) of often unknown function. In an effort to learn more about the function of fhe non- catalytic protein segments in these complex enzymes, we carried out a deletion study of the dorriains found in fhe T. maritima xyianase. Results Isolation, physical characterization, and sequence analysis of tiie xynA gene Recombinani E coH f^/I5219 clones from a T. maritima MSB8 gene libr'ary (Gabelsberger et a!.. 1993) in vector pSUI (Schrjiler ef ai, 1985) were screened for thermo- stable xyianase activity. For this purpose, several pools of clones (representing 750 clones each) wer'e made. Colonies were washed from replica plates and inoculated into 250 mi of Luria broth (LB) supplemented wifh ampicil- lin. After growth, ceil-free extracts of the pooled cells were pr-epared with lhe lysozyme Ireeze-thaw procedur'e and