A Specific, Robust, and Automated Method for Routine At-Line Monitoring of the Concentration of Cellulases in Genetically Modified Sugarcane Plants

Bagasse is one of the waste crop materials highlighted as commercially viable for cellulosic bio-ethanol production via enzymatic conversion to release fermentable sugars. Genetically modified sugarcane expressing cellobiohydrolases (CBH), endoglucanase (EG), and β-glucosidases (BG) provide a more cost-effective route to cellulose breakdown compared to culturing these enzymes in microbial tanks. Hence, process monitoring of the concentration profile of these key cellulases in incoming batches of sugarcane is required for fiscal measures and bio-ethanol process control. The existing methods due to their non-specificity, requirement of trained analysts, low sample throughput, and low amenability to automation are unsuitable for this purpose. Therefore, this paper explores a membrane-based sample preparation method coupled to capillary zone electrophoresis (CZE) to quantify these enzymes. The maximum enzyme extraction efficiency was obtained by using a polyethersulfone membrane with molecular cut-off of 10 kDa. The use of 15 mM, pH 7.75, phosphate buffer resulted in CZE separation and quantification of CBH, EG, and BG within 10 min. Migration time reproducibility was between 0.56% and 0.7% and hence, suitable for use with automatic peak detection software. Therefore, the developed CZE method is suitable for at-line analysis of BG, CBH, and EG in every batch of harvested sugarcane.