Production and characterization of thermophilic glycosyl- and glycoside hydrolases from Alicyclobacillus acidocaldarius

Thermophilic enzymes show better stability, better tolerance to contaminants and are often more active than their lower temperature counterparts.  Since 2006, the Idaho National Laboratory (INL) has been working with an industry partner to develop cellulose and hemicellulose degrading enzymes from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius (AA) for utilization in biofuels applications.  AA produces a suite of glycosyl- and glycoside hydrolases including endo-β-1, 4-glucanase (EG), endo-β-1, 4-xylanase (EX), β-glucosidase (BG), β-xylosidase (BX), accessory enzymes including α-L-arabinofuranosidase and α-glucuronidase, and several esterases.  Optimized genes were expressed in E. coli and the pH and temperature dependence of the recombinant proteins determined using proxy substrates.  Specific activities measured on natural substrates were benchmarked against commercially available enzymes.  The optimum pH and temperature for these enzymes were pH 6.0 and 70-80°C.  Activity was observed over the range 50-80°C and from pH 4-8.  The enzymes were benchmarked at two conditions, 50°C and pH 4.8 (fungal optima) and at 70°C and pH 6.0 against a BG purified from Novozymes TN188, a fungal BX, an EG purified from Accelerase and an EX purified from Multifect Xylanase.   The AA BG and BX showed 8-fold and 4-fold higher activity, respectively, at the fungal optima and both showed 16-fold higher activity at the AA optimum conditions.  The AA EG had activity similar to the fungal EG at the fungal optima, and 2.8-fold higher activity at the AA optimum.  The AA EX had about half the activity level of the fungal EX at the AA optimum.