Screening of cellulolytic bacteria from rotten wood of Qinling (China) for biomass degradation and cloning of cellulases from Bacillus methylotrophicus

Abstract

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.

Cloning and characterization of low-temperature adapted GH5-CBM3 endo-cellulase from Bacillus subtilis 1AJ3 and their application in the saccharification of switchgrass and coffee grounds

“…Bacillus subtilis 1AJ3 (GenBank No. MG062801), a cellulolytic bacterium isolated from rotten wood obtained from Qinling Mountains (China) (Ma et al 2020), which was used as the clone template in this study. An analysis of the 16S rRNA gene sequence of strain 1AJ3 shared 100% sequence identity with the 16S rRNA gene of B. subtilis strain JCM 1465, B. subtilis strain NBRC 13719, and B. subtilis strain DSM 10 in the NCBI (National Center for Biotechnology Information) database.…”

Succession of Gut Microbial Structure in Twin Giant Pandas During the Dietary Change Stage and Its Role in Polysaccharide Metabolism

“…Both of them may degrade fermentable fiber substrates in the panda (Williams et al, 2013;Xia et al, 2014) by expressing GH5 (Wierzbicka-Wo et al, 2019). When the cubs consumed supplementary foods and bamboo, only GH5_2 genes produced by Paenibacillus was still abundant in the gut microbes, which is a special GH5 subfamily with high activity and stable structure (Ma et al, 2020). Enterobacteriaceae, Escherichia, and other taxa increased genes encoding GH8 and GH9.…”

Optimization of the cultivation conditions of Bacillus licheniformis BCLLNF-01 for cellulase production

“…In addition, bacterial cellulases are often more effective catalysts and may also be less inhibited by the presence of material hydrolyzed [ 4 ]. Due to the high cost of production, there is a constant search for new producers of cellulases and improvement of culture conditions to maximize enzymatic synthesis [ [5] , [6] , [7] , [8] ]. The prospection of microbial enzymes in the marine environment has been an important strategy in the search for new cellulase producers.…”