High-level expression of recombinant thermostable β-glucosidase in Escherichia coli by regulating acetic acid

et al. 2020

BMC Biotechnol

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.

Section: Cellulase Of B Methylotrophicusmentioning

confidence: 59%

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

Lü

et al. 2020

BMC Biotechnol

“…For examples, β-1,3-1,4-glucanase from Bacillus subtilis 168 with a crude enzyme activity of 1366.48U/mL had highest sequence similarity of 94% with Bgl [54], and a beta-glucosidase with a similar Mw (27.35kDa) from Bacillus licheniformis showed enzyme activity of 67.34 U/mL [55]. Importantly, all the reported similar Bgls did not possesses a better enzyme activity than the recombinant Bgl in the present study, even the industrial optimized beta-glucosidase (560.4U/mL) [56] and other heterogeneous expressed β-1,4-glucosidase from bacteria (Paenibacillus sp. 5.8U/mL [57]) and fungi (Daldinia eschscholzii, 3.21 U/mg [58]; Daldinia eschscholzii, 25.13 U/mL [59]).…”

Section: Cellulase Of B Methylotrophicusmentioning

confidence: 58%

Screening of Cellulolytic Bacteria from Rotten Wood of Qinling (China) for Biomass Degradation and Cloning of Cellulases from Bacillus methylotrophicus

et al. 2019

Preprint

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.

“…For examples, β-1,3-1,4-glucanase from Bacillus subtilis 168 with a crude enzyme activity of 1366.48U/mL had highest sequence similarity of 94% with Bgl [54], and a beta-glucosidase with a similar Mw (27.35kDa) from Bacillus licheniformis showed enzyme activity of 67.34 U/mL [55]. Importantly, all the reported similar Bgls did not possesses a better enzyme activity than the recombinant Bgl in the present study, even the industrial optimized betaglucosidase (560.4U/mL) [56] and other heterogeneous expressed β-1,4-glucosidase from bacteria (Paenibacillus sp. 5.8U/mL [57]) and fungi (Daldinia eschscholzii, 3.21 U/mg [58]; Daldinia eschscholzii, 25.13 U/mL [59]).…”

Section: Cellulase Of B Methylotrophicusmentioning

confidence: 58%

Screening of Cellulolytic Bacteria from Rotten Wood of Qinling (China) for Biomass Degradation and Cloning of Cellulases from Bacillus methylotrophicus

et al. 2019

Preprint

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.