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      Identification of a Novel Fungus, Leptosphaerulina chartarum SJTU59 and Characterization of Its Xylanolytic Enzymes

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          Abstract

          Xylanolytic enzymes are widely used in processing industries, e.g., pulp and paper, food, livestock feeds, and textile. Furthermore, certain xylanotic enzymes have demonstrated the capability to improve the resistance and immunity of plants. Screening of high-yield microbial xylanolytic enzyme producers is significant for improving large-scale cost-effective xylanolytic enzyme production. This study provided new evidence of high-level xylanolytic enzyme production by a novel fungus, designated Leptosphaerulina chartarum SJTU59. Under laboratory conditions, L. chartarum SJTU59 produced xylanolytic enzymes of up to 17.566 U/mL (i.e., 878.307 U/g substrate). The enzyme solution was relatively stable over a wide range of pH (pH 3.0 to pH 9.0) and temperature (40°C to 65°C) while showing high resistance to the majority of metal ions tested. Composition analysis of the hydrolytic products of xylan showed sufficient degradation by xylanolytic enzymes from L. chartarum SJTU59, mainly the monosaccharide xylose, and a small amount of xylobiose were enzymatically produced; whereas in the presence of sufficient xylan substrates, mainly xylooligosaccharides, an emerging prebiotic used in food industry, were produced. In addition, the xylanolytic enzyme preparation from L. chartarum SJTU59 could initiate tissue necrosis and oxidative burst in tobacco leaves, which may be related to enhanced plant defense to adversity and disease. L. chartarum SJTU59 possessed a complex xylanolytic enzyme system, from which two novel endo-β-1,4-xylanases of the glycoside hydrolase (GH) family 10, one novel endo-β-1,4-xylanase of the GH family 11, and one novel β-xylosidase of the GH family 43 were obtained via rapid amplification of complementary DNA ends. Given the high yield and stable properties of xylanolytic enzymes produced by L. chartarum SJTU59, future studies will be conducted to characterize the properties of individual xylanolytic enzymes from L. chartarum SJTU59. xylanolytic enzymes-encoding gene(s) of potential use for industrial and agricultural applications will be screened to construct genetically engineered strains.

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          Most cited references13

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          Cellulase families revealed by hydrophobic cluster analysis.

          The amino acid sequences of 21 beta-glycanases have been compared by hydrophobic cluster analysis. Six families of cellulases have been identified on the basis of primary structure homology: (A) endoglucanases B, C and E of Clostridium thermocellum; endoglucanases of Erwinia chrysanthemi and Bacillus sp.; endoglucanase III of Trichoderma reesei; endoglucanase I of Schizophyllum commune; (B) cellobiohydrolase II of T. reesei; endoglucanases of Cellulomonas fimi and Streptomyces sp; (C) cellobiohydrolases I of T. reesei and of Phanerochaete chrysosporium; endoglucanase I of T. reesei; (D) endoglucanase A of C. thermocellum and an endoglucanase from Ce. uda; (E) endoglucanase D of C. thermocellum and an endoglucanase from Pseudomonas fluorescens; (F) xylanases of C. thermocellum and of Cryptococcus albidus and the cellobio-hydrolase of Ce. fimi. For each family, conserved potentially catalytic residues have have been listed and previous allocations of the active-site residues are evaluated in the light of the alignment of the amino acid sequences. A strong homology is also reported for the putative cellulose-binding domains of cellulases of Ce. fimi and of P. fluorescens.
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            Direct ethanol production from hemicellulosic materials of rice straw by use of an engineered yeast strain codisplaying three types of hemicellulolytic enzymes on the surface of xylose-utilizing Saccharomyces cerevisiae cells.

            The cost of the lignocellulose-hydrolyzing enzymes used in the saccharification process of ethanol production from biomass accounts for a relatively high proportion of total processing costs. Cell surface engineering technology has facilitated a reduction in these costs by integrating saccharification and fermentation processes into a recombinant microbe strain expressing heterologous enzymes on the cell surface. We constructed a recombinant Saccharomyces cerevisiae that not only hydrolyzed hemicelluloses by codisplaying endoxylanase from Trichoderma reesei, β-xylosidase from Aspergillus oryzae, and β-glucosidase from Aspergillus aculeatus but that also assimilated xylose through the expression of xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. The recombinant strain successfully produced ethanol from rice straw hydrolysate consisting of hemicellulosic material containing xylan, xylooligosaccharides, and cellooligosaccharides without requiring the addition of sugar-hydrolyzing enzymes or detoxication. The ethanol titer of the strain was 8.2g/l after 72h fermentation, which was approximately 2.5-fold higher than that of the control strain. The yield (grams of ethanol per gram of total sugars in rice straw hydrolysate consumed) was 0.41g/g, which corresponded to 82% of the theoretical yield. The cell surface-engineered strain was thus highly effective for consolidating the process of ethanol production from hemicellulosic materials. Copyright © 2011 Elsevier B.V. All rights reserved.
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              The enzymatic activity of fungal xylanase is not necessary for its elicitor activity.

              Fungal xylanases from Trichoderma spp. are potent elicitors of defense responses in various plants. To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei. For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S). Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity. Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity. In contrast, these mutated forms showed no comparable drop in elicitor activity. They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants. These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                9 September 2013
                : 8
                : 9
                : e73729
                Affiliations
                [1]School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China
                Russian Academy of Sciences, Institute for Biological Instrumentation, Russian Federation
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JC QW. Performed the experiments: QW. Analyzed the data: QW Yaqian Li MW TZ. Contributed reagents/materials/analysis tools: QW Yaqian Li Yingying Li SG. Wrote the paper: QW. English Polish: JC QW Yingying Li.

                Article
                PONE-D-13-17861
                10.1371/journal.pone.0073729
                3767624
                24040044
                2d44822e-e86c-466c-bb24-c3510232c371
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 1 May 2013
                : 20 July 2013
                Page count
                Pages: 14
                Funding
                This work was supported by China Agriculture Research System (No. CARS-02) and the National High Technology Research and Development Program of China (No. 2011AA10A205). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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