Expression of novel glucose tolerant β-glucosidase on cell surface by Rhodotorula glutinis isolate Vijaya Rani a , Sukanta Dash b , Lata Nain a , Anju Arora a,n a Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110012, India b Division of Design of Experiments, Indian Agricultural Statistical Research Institute, New Delhi 110012, India article info Article history: Received 30 March 2015 Received in revised form 6 June 2015 Accepted 8 June 2015 Keywords: β-glucosidase Rhodotorula glutinis Cell-surface expression Response surface methodology Box–Behnken design abstract Enzymatic hydrolysis is the most crucial step in bioconversion of lignocellulosic biomass to ethanol as efficient conversion of polymers to fermentable sugars determines final ethanol concentration. Cellulase complex used for commercial biomass hydrolysis is mostly derived from Trichoderma sp. and contains β- glucosidase o1% of total proteins, a ratio much lower than required for optimum saccharification. Therefore, supplementing cellulases with exogenous β-glucosidase having desired properties and bio- prospecting organisms producing such enzyme is an important activity. β-glucosidase producing yeast Rhodotorula glutinis was isolated from decaying vegetables. The β-glucosidase enzyme was constitutively expressed on the cell surface. Addition of surfactant to the culture medium or sonication could not release cell-associated β-glucosidase enzyme. While cellulose and glucose induced high levels of β- glucosidase activity, unusual stimulation of β-glucosidase production was observed with Cellobiose and Soybean meal additive in minimal medium. The enzyme had temperature optimum of 50 °C and pH 6.0– 6.5 and showed high glucose tolerance ability as 38.62% activity was retained even at 1.2 M glucose concentration. Culture medium for β-glucosidase production was optimised using Response Surface Methodology (RSM) with Box–Behnken design. The optimised predicted values for the three responses: extracellular enzyme activity – 0.048 IU/mL; extracellular specific enzyme activity – 0.649 IU/mg of protein; cell associated enzyme activity – 9.389 IU/mL were obtained. Thus, R. glutinis could be a po- tential gene source of β-glucosidase with desirable properties to be exploited in biomass hydrolysis. & 2015 Elsevier Ltd. All rights reserved. 1. Introduction Lignocellulose, a renewable organic material, consisting of cellulose, hemicellulose and lignin, forms the major structural component of all plant cells (Dashtban et al., 2009). It provides one of the cheapest and readily available feedstock for production of biofuels and other value added biomolecules which could help relieve the crisis in the highly energy exhaustive era and also lighten the pressure on the limited natural fossil fuel reserves. However, for production of ethanol from lignocellulosic biomass, various steps like pretreatment, hydrolysis, fermentation, separa- tion and purification are involved (Banerjee et al., 2010). Of all these steps, enzymatic hydrolysis is most crucial as efficient con- version of polymers to sugars determines efficient ethanol pro- duction (Del Pozo et al., 2012). Hydrolysis of the pretreated biomass is mainly carried out by cellulases which convert cellulose to glucose (Teugjas and Valjamae, 2013). For commercialization of cellulosic ethanol, it is important to get concentrated glucose slurries which demands higher cellulose loading and its efficient break down to glucose using cellulase enzymes. Cellulase complex consists mainly of exocellulases (EC 3.2.1.91), endocellulases (EC 3.2.1.4) and β-glu- cosidases (EC 3.2.1.21). Exocellulases and endocellulases create cellobiose and other oligomers which are acted upon by β-gluco- sidases to form sugar monomers. Low concentration of β-gluco- sidases not only results in the accumulation of cellobiose, but also inhibits the activity of the other two enzymes due to feed-back inhibition. Therefore, the major challenge in improving the effi- ciency of cellulose hydrolysis is overcoming the product inhibition of cellulases by cellobiose (Yue et al., 2004). Unfortunately, most of the cellulases used for commercial biomass hydrolysis, produced by saprophytic fungi mainly Tri- choderma sp. are deficient in β-glucosidases (Del Pozo et al., 2012; Wang et al., 2013). Therefore, supplementation of exogenous en- zyme in cellulase complex is often the chosen strategy to increase saccharification efficiency. Reports on improvement in overall cellulase performance upon addition of β-glucosidase are Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bab Biocatalysis and Agricultural Biotechnology http://dx.doi.org/10.1016/j.bcab.2015.06.004 1878-8181/& 2015 Elsevier Ltd. All rights reserved. n Corresponding author. Fax: þ91 11 25741648. E-mail address: anjudev@yahoo.com (A. Arora). Please cite this article as: Rani, V., et al., Expression of novel glucose tolerant β-glucosidase on cell surface by Rhodotorula glutinis isolate. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.06.004i Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎