Abstract
BackgroundThe complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. But endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), however, the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the next cellulose degradation. This will increase the cost of cellulose degradation.ResultsThe glucose-tolerant β-glucosidase (BGL) from Thermoanaerobacterium thermosaccharolyticum DSM 571 was fused with cellulose binding domain (CBD) of Clostridium cellulovorans cellulosome anchoring protein by a peptide linker. The fusion enzyme (BGL-CBD) gene was overexpressed in Escherichia coli with the maximum β-glucosidase activity of 17 U/mL. Recombinant BGL-CBD was purified by heat treatment and following by Ni-NTA affinity. The enzymatic characteristics of the BGL-CBD showed optimal activities at pH 6.0 and 65°C. The fusion of CBD structure enhanced the hydrolytic efficiency of the BGL-CBD against cellobiose, which displayed a 6-fold increase in V max /K m in comparison with the BGL. A gram of cellulose was found to absorb 643 U of the fusion enzyme (BGL-CBD) in pH 6.0 at 50°C for 25 min with a high immobilization efficiency of 90%. Using the BGL-CBD as the catalyst, the yield of glucose reached a maximum of 90% from 100 g/L cellobiose and the BGL-CBD could retain over 85% activity after five batches with the yield of glucose all above 70%. The performance of the BGL-CBD on microcrystalline cellulose was also studied. The yield of the glucose was increased from 47% to 58% by adding the BGL-CBD to the cellulase, instead of adding the Novozyme 188.ConclusionsThe hydrolytic activity of BGL-CBD is greater than that of the Novozyme 188 in cellulose degradation. The article provides a prospect to decrease significantly the operational cost of the hydrolysis process.
Highlights
The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase
Endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), but the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the cellulose degradation
Gene cloning and production of BGL-CBD fusion For the fusion of BGL with CBD, the plasmids pET-BGLCBD and pET-BGL-Linker-CBD were successfully constructed by linking the T. thermosaccharolyticum DSM 571 glucose-tolerant β-glucosidase gene bgl [12] to the gene fragment encoding CBD from C. cellulovorans cellulosome anchoring protein with or without a flexible peptide Linker (PPP)
Summary
The complete degradation of the cellulose requires the synergistic action of endo-β-glucanase, exo-β-glucanase, and β-glucosidase. Endo-β-glucanase and exo-β-glucanase can be recovered by solid–liquid separation in cellulose hydrolysis by their cellulose binding domain (CBD), but the β-glucosidases cannot be recovered because of most β-glucosidases without the CBD, so additional β-glucosidases are necessary for the cellulose degradation. This will increase the cost of cellulose degradation. As compared on the enzyme properties, the BGL showed higher tolerant to glucose and cellobiose, more efficient in hydrolysis of cellobiose, more thermal stability than β-glucosidases from other microorganisms [12]
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