Two different endoxylanase genes of deep biosphere bacteria were cloned and overexpressed in E. colt. Overexpression resulted in 11- and 8-fold improvement in endoxylanase activity of Xy1SG7 and Xy1SG13 proteins, respectively. Purified Xy1SG7 and Xy1SG13 showed an optimal catalytic temperature of 60 degrees C and 65 degrees C, respectively, with a half-life of 20 h under similar operational conditions (60 degrees C, pH 7.0). The KM value for Xy1SG7 was 0.2371 mg mL(-1) compared to 0.4768 mg mL(-1) exhibited by Xy1SG13 with beechwood xylan. Evaluation of surface characteristics of endoxylanases through Surface Plasmon Resonance highlights a novel approach to characterization of binding prior to covalent immobilization. For improved hydrolysis of corn stover (CS), two enzymatic cocktails were prepared by mixing immobilized Xy1SG7 and XyISG13 with Cellic (R) C-Tec2, separately. Application of purified immobilized endoxylanases for CS hydrolysis is a pioneering effort in bioethanol production. Both immobilized endoxylanase were successfully reused up to 4 hydrolysis cycles with a fresh supplement of Cellic C-Tec2 each time. Kluyveromyces mancianus yeast was selected for its thermotolerant properties, weak glucose repression and ability to metabolize the hemicellulolytic hydrolyzate. A novel high temperature-high-pressure (HTHP) technique was deployed for maximal utilization of sugars, and enhanced recovery of the produced ethanol, during fermentation. Intermittent use of HTHP simultaneously with the fermentation reaction resulted in 18.2% improved ethanol production over the conventional fermentation process. Simultaneous recovery of ethanol prompted the complete utilization of reducing sugars, compared to a residual concentration of 11.2 g/L observed with a conventional process. These findings are the first to be reported on the application of the HTHP technique for improved ethanol production, and on a highly thermostable endoxylanase showing the lowest KM value to date.