Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ tfosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/- 4.3] and 28.2[+/- 2.7] mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.