Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80 degrees C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80 degrees C.
Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5-7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.
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