The CBDs of a Thermostable Xylanase from Rhodothermus marinus
Eva NORDBERG KARLSSON, Eva BARTONEK-ROXA*, and Olle HOLST
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden
The xylanase encoding gene, xyn, from Rhodothermus marinus has been cloned and expressed in Escherichia coli. The gene comprises 5 different domains in an unusual combination. The cellulose binding domains (CBDs) encoded by xynl are repeated in tandem at the N-terminus and show similarity with the CBD family IV. xynl is the first example of a CBD family IV encoding gene in combination with a xylan hydrolyzing catalytic domain of the glycosyl hydrolase family 10 (1).
The two xyn/-CBDs are 88 % identical and separated by a short linker peptide. These putative CBDs show sequence similarities with family IV CBDs of cenC from Cellulomonas fimi (2). The cenC-CBDs are the only family IV CBDs whose adsorption properties have been investigated so far. The similarity level between these domains of the two different organisms is approximately 50% (25% identity), a rather low level that could indicate that our CBDs belong to a different subgroup within the family.
The adsorption properties of the R. marinus CBDs are currently investigated. Initial studies on insoluble cellulose have been made using the full-length xylanase. No adsorption was observed using Avicel, but significant adsorption could be shown using regenerated phosphoric acid-swollen cellulose (PASC). PASC is composed of antiparallelβ-1,4-glucan chains and is considered to be amorphous. Avicel, composed of microcrystallites embedded within an amorphous matrix, has a crystallinity of approximately 50%. Based on our results so far, we think that the xynl-CBDs only adsorb to amorphous cellulose, a feature also seen for the CBDs of cenC. Surprisingly, we could not see any adsorption to Avicel which also contains amorphous cellulose, but this could be due to the detection limit of our experiments.
Among the existing applications for xylanases, the use in the bleaching of pulps for paper manufacturing is of great environmental interest. Due to process conditions, enzymes functioning at high temperatures are desirable. Rhodothermus marinus secretes several very thermostable xylanolytic enzymes as well as other polysaccharide hydrolyzing enzymes (3, 4).
The presence of a CBD, associated to a carbohydrate hydrolysing enzyme, has in several cases been reported to enhance the efficiency in degradation of complex and/or insoluble substrates. Therefore, the presence of a CBD in combination with a
