Xylose is a sugar that is plentiful in lignocellulosic biomass, but is currently underused. Despite it being a potential carbon source for 2G ethanol production, the native yeast Sacharomyces cerevisiae cannot assimilate xylose. One possible way to overcome this restriction would be the previous isomerization of xylose to xylulose, catalyzed by the enzyme xylose isomerase (XI). Due to the unfavorable chemical equilibrium of conversion of 5-xylose to 1-xylulose, this route requires simultaneous isomerization and fermentation (SIF), in order to shift the equilibrium. The present work describes a study of ethanol production from xylose in a SIF process, using a biocatalyst consisting of XI immobilized on chitosan and subsequently co-immobilized with baker's yeast in Caalginate gel. The effects of biocatalyst composition (enzyme and yeast loads) and temperature were evaluated. The biocatalyst composition was varied using enzyme loads from 5 to 20% (w/v) and cell concentrations from 5 to 17% (w/v). Productivity and yield increased together with the yeast concentration, while selectivity increased with the enzyme concentration. For a biocatalyst consisting of 10% (w/v) of yeast (50 g. L-1) and 20% of enzyme (120 x 10(3) IU.L-1), 98% conversion was achieved within 11 h, providing a yield of 0.35 +/- 0.02 g. g(-1) productivity of 2.07 +/- 0.17 g. L-1.h(-1), and ethanol/xylitol selectivity of 2.42 +/- 0.01. The temperatures tested were 32, 35, and 37 degrees C, and ethanol yield and productivity were around 0.35 g. g(-1) and 2.03 g .L-1.h(-1), respectively, in all the experiments. Although higher temperatures favor XI activity, 35 degrees C was selected because it favored ethanol formation.