Optimization of cellulose enzymatic hydrolysis is crucial for cost effective bioethanol production from
lignocellulosic biomass. Enzymes involved in cellulose hydrolysis are often inhibited by their
end-products, cellobiose and glucose. Efforts have been made to produce more efficient enzyme variants
that are highly tolerant to product accumulation; however, further improvements are still necessary.
Based on an alternative approach we initially investigated whether recently formed glucose could be
phosphorylated into glucose-6-phosphate to circumvent glucose accumulation and avoid inhibition of
beta-glucosidase from Bacillus polymyxa (BGLA). The kinetic properties and structural analysis of BGLA
in the presence of glucose-6-phosphate (G6P) were investigated. Kinetic studies demonstrated that
enzyme was not inhibited by G6P. In contrast, the presence of G6P activated the enzyme, prevented beta
glucosidase feedback inhibition by glucose accumulation and improved protein stability. G6P binding
was investigated by fluorescence quenching experiments and the respective association constant indicated
high affinity binding of G6P to BGLA. Data reported here are of great impact for future design strategies
for second-generation bioethanol production.