Xylan degradation by the thermophilic bacterium Rhodothermus marinus. Characterization and function of a thermostable xylanase

Abstract: A gene encoding a multidomain xylanase from the thermophilic bacterium Rhodothermus marinus has been cloned, sequenced and produced in Escherichia coli . The gene product was found to be a 109 kDa protein consisting of five domains. The catalytic domain and the two polysaccharide binding domains (PBD, previously called carbohydrate binding domains) have been cloned and produced separately for functional studies. The catalytic domain is classified under glycosyl hydrolase family 10, a family in which 3D-structure determined representatives exhibit 8-fold alpha/beta(TIM)-barrels. It catalyses endo-hydrolysis of xylans and to a lower extent mixed linkage (1-3,4) ß-glucans. Hydrolysis has been observed using oligosaccharides with a degree of polymerisation of 4 or higher. The two polysaccharide binding domains are likely displaying folds based on ß-strands. Their binding specificities to soluble substrates are corresponding well to observed substrate specificity of the catalytic domain. Among insoluble substrates they display binding to both amorphous cellulose and xylan. Both the binding level and thermal stability were markedly increased by additions of calcium ions. The three functionally characterized domains have transition temperatures for unfolding above, or long half-lives within, the growth temperatures (55-77 °C) of the organism R. marinus. The efficiency in enzyme aided bleaching of kraft pulps was tested for three catalytically active forms of the enzyme. All forms lead to brightness gains although at different extents. The gain was found to be dependent on both enzyme construction and pulp characteristics. The trial with an enzyme construct lacking the two N-terminal PBDs was most successful. A strategy for production in E. coli was also developed for two enzyme forms. The results show that production in the selected host-vector system was dependent on both cultivation mode, medium composition and type of inducer. Most efficient production was obtained in fed-batch cultivations in the presence of complex nutrients. Under these conditions the two alternative inducers, IPTG/lactose, resulted in approximately equal amounts of the produced recombinant proteins.