Background Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. Results In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15±18.94 U/mL and 0.130±0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. Conclusions Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.

Background: Biomass lignocellulose provide abandon cellulosic material for bioenergy. The synergy effect of the cellulase system still needs more attention to make it more efficient. Exocellulase was less of studies in former studies, especially recombinant exocellulase from bacteria. Results: An exocellulase Cbh -A from strain B. subtilis 1AJ3 was cloned and expressed in E.coli BL21. By sequence analysis, Cbh -A contains two domains: FlgJ and SH3_8. Enzymatic properties of Cbh -A were characterized, and the maximum enzyme activity was gotten under the condition of pH 6.4 and 50℃. Moreover, the enzyme reached the highest enzyme activity and kept stable under 50℃ for 4 h. Ion metal Mn 2+ had an activating effect on Cbh -A, while Cu 2+ as well as chemical EDTA had inhibition effect. Cbh -A had widely substrates specification that it could hydrolyze β-1,3-1,4/β-1,3/β-1,4 glucan linkage, α-1,4 glucan linkage . When it took cooperation with recombinant endocellulase and β-glucosidase also from the strain B. subtilis 1AJ3, synergism effect was observed through DS (degree of synergism) value by hydrolyzing CMC-Na, Avicel, and filter paper. At the same time, the synergism effect was also examined in biomass lignocellulose by comparing the saccharification rate and DS value of each material. Conclusions: Cbh -A contained a FlgJ domain and first studied as an exocellulase cloned from B. subtilis 1AJ3. The biochemical, enzymatic properties, ion metal, and substrate specificity of Cbh -A were analyzed, and a special character of Cbh -A could keep maximum activity after pre-warm at 50℃ for 4h. Cbh-A had good synergy effect with endocellulase and β-glucosidase from the same strain in variety kind of cellulosic substrates, including simple and complex biomass cellulose. It laid a foundation of application by mixed cellulases via synergy effect, and supply new thought for the degradation of biomass lignocellulose.

Background Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. Results In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15±18.94 U/mL and 0.130±0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. Conclusions Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and Bacillus methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from Bacillus methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.