The use of the hemicellulose fraction of biomass may be important for the feasibility of the production of second generation bioethanol. Wild strains of Saccharomyces cerevisiae are widely used in industry for production of 1st generation ethanol, and the robustness of this yeast is an important advantage in large scale applications. Isomerization of xylose to xylulose is an essential step in this process. This reaction is catalyzed by glucose isomerase (GI). A new biocatalyst is presented here for the simultaneous isomerization and fermentation (SIF) of xylose. Cl from Streptomyces rubiginosus was immobilized in chitosan, through crosslinking with glutaraldehyde, and the support containing the immobilized Cl (IGI-Ch) was co-immobilized with S. cerevisiae, in calcium alginate gel. The immobilization experiments led to high immobilized protein loads (30-68 mg x g(support)(-1)), high yields (circa of 100%) and high recovered enzyme activity (>90%). The IGI-Ch derivative with maximum activity presented 1700 IU x g(catalyst)(-1), almost twice the activity of a commercial immobilized GI, GENSWEET (R) IGI-HF. At typical operational conditions for xylose SIF operation (pH 5, 30-35 degrees C, presence of nutrients and ethanol concentrations in the medium up to 70 L-1), both derivatives, IGI-Ch and GENSWEET (R) IGI-HF retained app. 90% of the initial activity after 120 h, while soluble GI was almost completely inactive at pH 5, 30 degrees C. The isomerization xylose/xylulose, catalyzed by IGI-Ch, reached the equilibrium in batch experiments after 4 h, with 12,000 IU x L-1 (7 g(der) x L-1), at pH 5 and 30 degrees C, in the presence of fermentation nutrients. After co-immobilization of IGI-Ch with yeast in alginate gel, this biocatalyst succeeded in producing 12 g x L-1 of ethanol, 9.5 g x L-1 of xylitol, 2.5 g x L-1 of glycerol and 1.9 g x L-1 of acetate after consumption of 50 g x L-1 of xylose, in 48 h, using 32.5 x 10(3) IU x L-1 and 20 g(yeast) X L-1, at 35 degrees C and initial pH 5.3. (C) 2011 Elsevier Inc. All rights reserved.