Expression, purification and use of the soluble domain of Lactobacillus paracasei b-fructosidase to optimise production of bioethanol from grass fructans C.M. Martel a , A.G.S. Warrilow a , C.J. Jackson a , J.G.L. Mullins a , R.C. Togawa c , J.E. Parker a , M.S. Morris b , I.S. Donnison b , D.E. Kelly a , S.L. Kelly a, * a Institute of Life Science and School of Medicine, Swansea University, Swansea SA2 8PP, Wales, UK b Institute of Biological, Environmental & Rural Sciences, Aberystwyth University, Gogerddan, Aberystwyth SY23 3EB, Wales, UK c Embrapa Recursos Genéticos e Biotecnologia, Laboratório de Bioinformática, Parque Estação Biológica – Final W5 norte, Caixa Postal 02372, Brasília, DF 70770-900, Brazil article info Article history: Received 15 October 2009 Received in revised form 13 January 2010 Accepted 19 January 2010 Available online 12 February 2010 Keywords: Bioethanol Fructan Fructanase Lactobacillus paracasei Perennial ryegrass abstract Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lacto- bacillus paracasei b-fructosidase was overexpressed as truncated cytosolic protein ( t fosEp) in Escherichia coli. Purified 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 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 bioavail- able fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer’s yeast and GJ + 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. Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. 1. Introduction The use of plant biomass for the production of carbon–neutral biofuels continues to attract investment from research and com- mercial sectors (Larsen et al., 2008; Schmer et al., 2008). Industri- ally viable strategies to optimise the release of energy from cellulosic and lignocellulosic fractions of plant material (Hendriks and Zeeman, 2009 for recent review) continue to constitute a focal point for biofuel research (e.g. bioethanol from lignocellulosic bio- mass; Larsen et al., 2008). However, consideration of the complete spectrum of plant biomass that could be used for the production of bioethanol highlights additional reservoirs of carbon-energy; plant fructans comprise one of these sources. In addition to sucrose and starch, fructans contribute to the pool of storage carbohydrates in plants (Ritsema and Smeekens, 2003). The simplest plant fructan is inulin which consists of a linear chain of b(2-1)-linked fructose monomers that extends from the fructo- syl residue of a sucrose (a-b-1-2 linked glucose and fructose) star- ter molecule common to all plant fructans anabolised in vivo. Levan-type fructans also comprise a linear chain of fructose mono- mers; however these are linked via b(2-6) bonds. Finally, mixed- type fructans (gramminans) contain both b(2-1) and b(2-6)-linked fructose chains which can branch from the fructosyl and/or gluco- syl residues of the sucrose starter molecule. Several fructan-con- taining plant crops including white clover (Trifolium repens), dandelion leaves (Taraxacum spp.), perennial ryegrass (Lolium per- enne L.) and Jerusalem artichoke (Helianthus tuberosus) have been identified (see Kyazze et al., 2008). However, to date, few reports have been published on the production of bioethanol from plant fructans; those that are available tend to concentrate on the util- isation of H. tuberosus biomass (Nakamura et al., 1996; Szambelan et al., 2004). For example, it has been shown that inulin-type fruc- tans derived from Jerusalem artichoke can be converted to ethanol by acidic hydrolysis followed by fermentation with Saccharomyces cerevisiae or via direct fermentation using Kluyveromyces marxianus strains (Negro et al., 2006). Similar research with a wider range of fructan-containing plant substrates is now required. Given the total area (some 14 million hectares) of agricultural grassland in the UK (DEFRA, 2006), it is not surprising that peren- nial ryegrass has recently been identified as a possible substrate for the production of biofuels (Martinez-Perez et al., 2007). Unlike more specialist crops (e.g. wheat, sugar-beet) currently used for bioethanol production in Europe (Balat, 2007), ryegrass requires relatively few energy inputs (Donnison et al., 2009). Moreover, be- cause of its fast establishment and robustness, ryegrass can be cul- tivated in marginal areas – an important consideration given the concerns surrounding use of arable land for non-food crops. The 0960-8524/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.01.084 * Corresponding author. Tel.: +44 1792 292207; fax: +44 1792 503430. E-mail address: s.l.kelly@swansea.ac.uk (S.L. Kelly). Bioresource Technology 101 (2010) 4395–4402 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech