Expression, purification and use of the soluble domain of Lactobacillus paracasei b-fructosidase to optimise production of bioethanol from grass fructans

• Published in: Bioresource technology 2010-06, Vol.101 (12), p.4395-4402
• Main Authors: Martel, C M, Warrilow, AGS, Jackson, C J, Mullins, JGL, Togawa, R C, Parker, JE, Morris, MS, Donnison, I S, Kelly, DE, Kelly, S L
• Format: Article
• Language: English
• Subjects: Escherichia coli; Lactobacillus paracasei; Saccharomyces cerevisiae
• ISSN: 0960-8524
• DOI: 10.1016/j.biortech.2010.01.084

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei b-fructosidase was overexpressed as truncated cytosolic protein ( sub(t)fosEp) in Escherichia coli. Purified sub(t)fosEp was thermostable (10-50 C) with a pH optimum of 5; it showed highest affinity for bacterial levan (b[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (b[2-1] linkages) and sucrose (K sub(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with sub(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 + sub(t)fosEp 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 b-fructosidase from L. paracasei.