Utilization of Bacillus subtilis cells displaying a glucose-tolerant β-glucosidase for whole-cell biocatalysis

The microbial production of industrial enzymes requires a large number of complex biochemical steps for purification which increases their production cost. Additionally, poor thermo-stability of the purified enzymes under the operational conditions along with the challenges in their recovery and subsequent reuse, limit their usage in an industrial bioprocess. Surface display of heterologous enzymes on bacterial cells appear to be a suitable alternative. Bacillus subtilis, the most well characterized Gram-positive bacterium, is being increasingly studied as a host for surface display. We displayed a glucose-tolerant β-glucosidase (UnBgl1A) on the surface of B. subtilis cells using CWB_b as the anchor protein. These cells displaying UnBgl1A (SD-01) were directly employed for biocatalysis without cell lysis and enzyme purification. The SD-01 cells elicited ∼2 times more catalytic activity compared to the cells expressing the enzyme intracellularly (IN-01). The displayed enzyme and the purified enzyme elucidated similar glucose tolerance (IC₅₀ ∼0.9 M glucose), temperature optima (∼50 °C), and pH optima (∼6.0). The surface displayed UnBgl1A retained ∼50% activity after 4 h when stored at 50 °C whereas the purified UnBgl1A lost all its activity by the 4^th hour. Additionally, the SD-01 cells could be efficiently reused for 3 sets of reactions. Further, supplementation of a cellulase cocktail with the cells of the SD-01 strain resulted in ∼2 times more glucose release from sugarcane bagasse compared to supplementation with the purified UnBgl1A. Therefore, displaying enzymes on the B. subtilis cell surface could be an attractive platform for the commercial production of industrial enzymes.