34 kDa
2.4 Conclusions
This study reported for the first time the enhanced production of recombinant C. thermocellum hydrolytic GH5 cellulase and GH43 hemicellulase (α-L- arabinofuranosidase) in repetitive batch mode in different media. For GH5 cellulase production, in LB medium with batch mode, the enzyme activity, protein concentration and the cell OD were 2.2 U mg-1, 0.18 mg mL-1 and 1.4. A cell OD (2.8), protein concentration (0.37 mg mL-1) and enzyme activity (4.5 U mg-1) with an increment of 2-fold was observed in LB medium with repetitive batch mode as compared to batch mode. The cell free extract of recombinant GH5 cellulase displayed a 35 kDa band on SDS-PAGE. Zymogram study confirmed the cellulase activity of GH5. In case of terrific broth (TB) with batch mode, the enzyme activity of 2.4 U mg-1, protein concentration of 0.20 mg mL-1 and the cell OD of 4.8 was obtained. On the other hand, in repetitive batch mode, the enzyme activity, protein concentration and cell OD achieved was 5.0 U mg-1, 0.40 mg mL-1 and 7.5 respectively. Thus, a 2-fold increment both in activity of the enzyme and protein concentration along with 1.6-fold rise in cell biomass was gained in repetitive batch mode as compared with batch mode. Thus, as compared to LB medium in batch and repetitive batch modes, a 3.4- and 2.7-fold augmentation in cell biomass were obtained for TB in respective modes. In batch mode LB medium with glucose, the recombinant GH5 cellulase activity of 2.8 U mg-1 with protein concentration of 0.37 mg mL-1 and highest cell OD of 6.0 was obtained. The repetitive batch mode yielded the specific activity (5.7 U mg-1), protein concentration (0.45 mg mL-1) and cell OD of 9.6. Thus, a 4.2-fold and 3.4-fold escalation in cell biomass was obtained in LB
medium with glucose as a co-substrate in batch and repetitive batch modes as compared to LB medium without glucose in both modes.
For GH43 hemicellulase (α-L-arabinofuranosidase) production, the batch mode LB medium yielded the enzyme activity, protein concentration and the cell OD of 1.9 U mg-1, 0.20 mg mL-1 and 1.6, respectively. The repetitive batch mode of LB medium gave an enzyme activity of 3.0 U mg-1, protein concentration of 0.26 mg mL-
1 and cell OD of 2.5. A 1.6-fold augmentation both in enzyme activity and cell biomass was obtained in repetitive batch mode as compared with batch mode. The cell free extract of recombinant GH43 hemicellulase (α-L-arabinofuranosidase) displayed a 34 kDa band on SDS-PAGE. Zymogram study confirmed the hemicellulase activity of GH43. In terrific broth (TB) with batch mode, the enzyme activity, protein concentration and cell OD obtained were 2.0 U mg-1, 0.21 mg mL-1 and 3.7, respectively. Whereas in repetitive batch mode an enzyme activity (3.4 U mg-
1), protein concentration (0.28 mg mL-1) and cell OD (5.9) displaying around 2-fold increase in enzyme activity was obtained as compared with batch mode. In batch mode LB medium with glucose, GH43 hemicellulase (α-L-arabinofuranosidase) activity of 2.2 U mg-1 with protein concentration of 0.24 mg mL-1 and cell OD of 5.2 was obtained. Finally, the repetitive batch mode yielded specific activity (3.7 U mg-1), protein concentration (0.32 mg mL-1) and cell OD of 8.1. Thus, a 3.2-fold escalation in cell biomass was obtained in LB medium with glucose as a co-substrate in batch and repetitive batch modes as compared to LB medium without glucose in both modes. The enhanced production of recombinant enzymes in LB medium supplemented with glucose will aid in effective saccharification of various
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Chapter 3
Selection of cellulose and hemicellulose rich substrates and efficient pretreatment process for bioethanol production