Enzymatic properties of Thermoanaerobacterium thermosaccharolyticum β-glucosidase fused to Clostridium cellulovorans cellulose binding domain and its application in hydrolysis of microcrystalline cellulose.

The 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. The 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 Vmax/Km 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. The 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.