生物能源是未来最有可能取代化石能源的能源,纤维素生物质的降解仍需引起更多关注,和更可评价的纤维素分解菌分离将为这一领域奠定基础。秦岭具有独特的生物多样性,作为纤维素降解细菌的有希望的来源,表现出值得注意的特性。因此,这项工作的目的是寻找潜在的纤维素分解细菌,并验证从选定的强大细菌中克隆纤维素酶的可能性。
在本研究中,从秦岭腐烂木材中筛选并鉴定了55种潜在的纤维素分解菌。通过对不同纤维素底物的纤维素酶活性及降解效果的研究,进一步应用嗜甲基芽孢杆菌1EJ7、枯草芽孢杆菌1AJ3和枯草芽孢杆菌3BJ4水解小麦秸秆,玉米秸秆和柳枝草,结果表明,嗜甲基芽孢杆菌1EJ7是最主要的细菌,这也表明芽孢杆菌是腐烂木材中主要的纤维素分解菌。此外,扫描电子显微镜(SEM)和X射线衍射分析小麦秸秆的微观形貌和结晶度也证实了显着水解。在确定纤维素酶β-葡萄糖苷酶(243aa)和内切葡聚糖酶(499aa)的目标序列的基础上,成功地从甲基营养芽孢杆菌1EJ7中进行了异源克隆和表达,对纤维素降解具有良好的效果,酶活性为1670.15±18.94U/mL和0.130±0.002U/mL。分别。此外,基于氨基酸序列的分析,发现β-葡萄糖苷酶属于GH16家族,内切葡聚糖酶由GH5家族催化结构域和CBM3家族的碳水化合物结合模块组成。
根据筛选结果,秦岭腐烂木材中纤维素分解菌的鉴定及降解效果评价,它发现芽孢杆菌是分离菌株中的优势种,和甲基营养芽孢杆菌1EJ7在纤维素降解方面表现最好。同时,首次成功克隆并表达了嗜甲基芽孢杆菌的β-葡萄糖苷酶和内切葡聚糖酶,为纤维素降解及其在工业上的应用研究提供了菌株和重组酶的新材料。
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.
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.
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.