5-74: Processive endoglucanase, Cel9A, from Bacillus licheniformis features important and desirable properties for industrial application of cellulose hydrolysis

A Bacillus licheniformis endoglucanase, Cel9A, was recombinantly produced via secretion in Bacillus subtilis WB800, an 8-protease deficient strain, with a production yield of 50 mg/L. This 70-kDa enzyme has a bimodular structure with a GH9 catalytic module and a CBM3c carbohydrate-binding module. It is active towards CMC, ASC, Avicel, filter paper and pre-treated wood substrates. Our study illustrates that the CBM3c module has dual functions. The removal of CBM3c module of Cel9A abolishes its activities towards crystalline cellulose including Avicel and filter paper and its thermal stability. The truncated Cel9A (with CBM3c removed) is only active between 20-50°C with maximum activity at 37°C and is stable below 30°C. Cel9A has several features to be an attractive industrial enzyme for cellulose hydrolysis. It is active in a wide range of pH (pH 6-9 with optimal activity around pH 7-8) and temperature (20-80°C with maximum activity at 65°C). Most importantly, it retains >80% activity even when it has been incubated at 50°C for 2 days. This is an important and desirable feature since a common practice of cellulose hydrolysis nowadays is to enzymatically digest the pretreated biomass at 50°C for 2 days. Furthermore, Cel9A is a processive endoglucanase. This is another vital feature needed for efficient hydrolysis of crystalline substrates. It can release 82% of soluble reducing sugars and 18% of insoluble reducing sugars from filter paper. In terms of its modes of action, Cel9A acts on the cellulose chain from the non-reducing end and releases cellotetraose as the major end product.