Original Research Paper Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat bran as carbon source Cynthia Barbosa Rustiguel, João Atílio Jorge, Luis Henrique Souza Guimarães n Departamento de Biologia – Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, 14040-901 Ribeirão Preto, São Paulo, Brazil article info Article history: Received 5 February 2015 Received in revised form 9 April 2015 Accepted 22 May 2015 Keywords: Invertase Fructooligosaccharide Enzyme purification Sucrose abstract The filamentous fungus Aspergillus phoenicis (Aspergillus saitoi) produced high levels of mycelial β-D- fructofuranosidase (invertase) when cultivated under submerged fermentation using Khanna medium with wheat bran as the carbon source for 72 h at 40 °C, under orbital agitation (100 rpm). The mycelial invertase was purified 20-fold with 24% recovery through two chromatographic steps (DEAE-cellulose and Sephacryl S-200). The enzyme was characterized as a monomeric glycoprotein with 2% carbohydrate content and a native molecular mass of 131 kDa comprising two 70-kDa subunits. The optimal tem- perature and pH for activity were 65 °C and 4.5, respectively. The enzyme was resistant to temperatures of 50 °C and 60 °C and stable at pH 4.0–7.0. Activity increased in the presence of different ions, especially Mn 2 þ ( þ177 %), and Ag þ increased the invertase activity by 91%. The mycelial invertase hydrolyzed sucrose, raffinose, and inulin, with greater specificity for the former. The K 1/2 and V max values for sucrose were 22.5 mM and 124.9 U mg 1 , respectively. & 2015 Published by Elsevier Ltd. 1. Introduction Invertases (β-D-fructofuranosidase, EC 3.2.1.26) are enzymes that catalyze the breakdown of β-2,1 glycosidic bonds from su- crose molecule to obtain an equimolar mixture of D-glucose and D-fructose known as invert sugar (Álvaro-Benito et al., 2007). These enzymes can be grouped as acid, neutral, and alkaline in- vertases based on the pH of maximal activity (Winter and Huber, 2000). Neutral and alkaline invertases have been reported in plants such as Arabidopsis thaliana (Xiang et al., 2011) and cya- nobacteria such as Anabaena sp. (Vargas and Salerno, 2010). Comparatively, acid invertases are found in plants and micro- organisms such as bacteria and fungi (BRENDA – The Compre- hensive Enzyme Information System). Invertase production by fi- lamentous fungi has been described in Cladosporium cladospor- ioides (Almeida et al., 2005), Aspergillus niger (Reddy et al., 2010), Aspergillus caesiellus (Novaki et al., 2010), Termitomyces clypeatus (Chowdhury et al., 2009), and A. phoenicis (Rustiguel et al., 2010), among others. Fungal invertases have attracted interest for various industrial applications, including those in the food and beverage industry. The invert sugar syrup obtained from sucrose hydrolysis by fungal invertases is sweeter than sucrose and does not crystallize at a high concentrations (Bayramoglu et al., 2003). In addition, some microbial fructofuranosidases catalyze the transfructosylating re- action at high sucrose concentrations ( Z20%) to obtain fructoli- gosaccharides (FOS) such as 1-kestose, nystose, and fructosyl nystose (Guimarães, 2012). The first step to determine the potential applications of a specific enzyme is to investigate its physico-chemical properties. If important and unique properties are observed, further genetic engineering studies are conducted to improve the production and/ or enzyme properties. The extracellular fructofuranosidase from A. phoenicis was previously characterized and showed interesting and intriguing properties (Rustiguel et al., 2010). However, to our knowledge, the mycelial fructofuranosidase has not been pre- viously investigated. Herein, this manuscript describes the pro- duction and characterization of a mycelial fructofuranosidase produced by A. phoenicis under submerged fermentation using wheat bran as the carbon source. 2. Material and methods 2.1. Microorganism and culture conditions The filamentous fungus A. phoenicis (A. saitoi) was maintained Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bab Biocatalysis and Agricultural Biotechnology http://dx.doi.org/10.1016/j.bcab.2015.05.004 1878-8181/& 2015 Published by Elsevier Ltd. n Corresponding author. Fax: þ55 16 3602 3668. E-mail address: lhguimaraes@ffclrp.usp.br (L.H.S. Guimarães). Please cite this article as: Rustiguel, C.B., et al., Characterization of a thermo-tolerant mycelial β-fructofuranosidase from Aspergillus phoenicis under submerged fermentation using wheat.... Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.05.004i Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎