Electronic Journal of Biotechnology 18 (2015) 464–470 Contents lists available at ScienceDirect Electronic Journal of Biotechnology Research Article Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation Larissa Serrani Valera a, João Atílio Jorge b, Luis Henrique Souza Guimarães b,⁎ a Instituto de Química de Araraquara, Universidade Estadual Paulista “Júlio de Mesquita Filho”, Rua Prof. Mario Degni s/n, Quitandinha, 14800-900 Araraquara, São Paulo, Brazil b 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 abstract Article history: Background: Tannases are enzymes with biotechnological potential produced mainly by microorganisms as Received 30 June 2015 filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Accepted 29 September 2015 Aspergillus carbonarius is described. Available online 28 October 2015 Results: The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent Keywords: for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The Aspergillus second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. Microbial enzymes Solid-state fermentation The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated Tannase through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate Tannic acid content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20–60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion: A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application. ©2015Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 1. Introduction as hydrolysis products [2]. Gallic acid is an important intermediary in the synthesis of the antibacterial drug trimethoprim, which is used in Tannins are polyphenols found in different plant species. They are the pharmaceutical and food industries. Gallic acid is also used as a often found in the bark, root leaf, wood, seed and fruit of plants. These precursor in the chemical and enzymatic synthesis of the antioxidant molecules are able to form complexes with proteins, digestive propyl gallate. Tannases can also be used in the beverage industry in enzymes, starch and minerals. As a result, tannins are characterized as the clarification processes of beer, fruit juices, instant tea and wines, as toxic, anti-nutritional agents that reduce digestibility and protein well as in effluent treatment performed in the leather industry and for availability in ruminants. In general, tannins can be separated in two agro-industrial wastes [3]. Despite its applicability and importance, major groups: condensed tannins and completed tannins. The former the practical use of tannases is limited. is difficult to hydrolyze while the latter is not. Hydrolysable tannins The main sources of tannases are microorganisms such as bacteria, are composed by a polyol (mainly glucose) as a central core esterified yeast and filamentous fungi. Among the filamentous fungi, Aspergillus by gallic acid, digallic acid (gallotannins), or ellagic acid (ellagitannins) and Penicillium are important tannase producers [4]. Recently, the [1]. production and characterization of fungal tannases have received The enzymatic hydrolysis of these hydrolysable tannins is achieved significant scientific attention, aimed at understanding their by the action of the tannin acyl hydrolyze (EC 3.1.1.20), also known as biological function, mechanism of action, biotechnological potential tannase. This enzyme catalyzes the breakdown of the ester and and applicability. Fungal tannase can be produced by both depsidic bonds found in tannic acid to generate gallic acid and glucose submerged fermentation [5] and solid-state fermentation (SSF) methods using agro-industrial residues as carbon sources or substrates [4,6]. Thermo-tolerant and solvent tolerant tannases are ⁎ Corresponding author. E-mail address: [email protected] (L.H.S. Guimarães). considered important enzymes with biotechnological potential Peer review under responsibility of Pontificia Universidad Católica de Valparaíso. [5,7]. Although both the production of tannase from fungal sources http://dx.doi.org/10.1016/j.ejbt.2015.09.008 0717-3458/© 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. L.S. Valera et al. / Electronic Journal of Biotechnology 18 (2015) 464–470 465 Table 1 Pernambuco, Brazil, according to morphological characteristics, and Influence of substrate/carbon source on tannin acyl hydrolase from A. carbonarius deposited in the culture collection of the Laboratory of Microbiology under SSF. from the Faculty of Philosophy, Science and Letters, University of São Substrate Enzymatic activity Paulo, Brazil. The microorganism was maintained in PDA slants stored (U/g of substrate) at 4°C and new cultures were obtained in 30-d intervals. Sugar cane bagasse 0.0 SSF was achieved using 5 g of different plant leaves and Wheat 0.56 ± 0.02 agro-industrial residues as substrate/carbon sources in 25 mL Leaves from A. occidentale 1.59 ± 0.01 Erlenmeyer flasks and humidification (1:1 w/v) with distilled water, Leaves from Coffea arabica 1.0 ± 0.01 Leaves from Camellia sinensis 3.60 ± 0.02 tap water or salt solutions (SR salt solution [8], Khanna salt solution [9], Leaves from Eucalyptus sp. 0.50 ± 0.01 Vogel salt solution [10]). The culture media was autoclaved at 120°C for Leaves from M. indica 0.1 ± 0.03 25 min and 1.5 atm. A spore suspension (2 mL of 105 spore/mL) was Leaves from M. esculenta 2.74 ± 0.04 used to inoculate the media. Fungal growth was conducted at 30°C for Linseed 0.70 ± 0.01 different periods depending on the experiment. Crushed corn 0.0 Sorghum 0.10 ± 0.01 2.2. Enzyme extraction and its characterization have been investigated, further studies are The crude extract containing tannin acyl hydrolase was obtained required to identify new tannase sources with novel attractive with the addition of 25 mL of cold distilled water. The mixture was properties. The purpose of the present study was therefore to agitated with a magnetic stirrer for 20 min at 4°C. Cultures were then present Aspergillus carbonarius as a promising source of tannase harvested through vacuum filtration using gauze and Whatman filter with interesting properties for biotechnological application. paper No. 1. The free cell extract obtained was dialyzed in distilled water for 24 h at 4°C and used for determining enzymatic activity and 2. Material and methods for purification. 2.1. Microorganism and culture conditions 2.3. Analysis of enzymatic activity The filamentous fungus A. carbonarius was isolated from soil and Tannin acyl hydrolase activity was determined using methanolic identified by the Laboratory of Microbiology, Federal University of rhodanine as described by Sharma et al. [11] with modification: 0.2% Fig. 1. (a) Tannase production by the fungus A. carbonarius under SSF using different moisture agents; (b) the effect of different proportions of tap water as the moisture agent on tannase production; (c) the influence of the incubation period on tannase production. The same case letter indicates that there is no significant statistical difference among the media (p = 0.05). 466 L.S. Valera et al. / Electronic Journal of Biotechnology 18 (2015) 464–470 Fig. 2. (a) DEAE-Cellulose and (b) Sepharose CL-6B chromatographic profiles for tannase produced by the fungus A. carbonarius. tannic acid, the natural substrate for tannases, in 100 mM sodium above. The standards used were β-amylase (200 kDa), alcohol acetate buffer pH 5.0. The mixture of the reaction was constituted dehydrogenase (150 kDa), bovine serum albumin (66 kDa) and with 250 μL of substrate solution and 250 μL of enzymatic extract. The carbonic anhydrase (29 kDa). The void of 86.3 mL was determined reaction was conducted at different temperatures and periods, using blue dextran. The denatured molecular mass was determined by depending on the experiment, and was finalized by adding of 300 μL 6% SDS-PAGE [14]. The protein bands were stained as previously of a methanolic rhodanine solution 0.667% (m/v). After 5 min, 200 μL published [15] using Coomassie Blue Silver G-250. α-Macroglobulin of 0.5 N KOH were added, followed by 4 mL of distilled water. (169 kDa), β-galactosidase (112.5 kDa), lactoferrin (92 kDa), pyruvate Absorbance was then measured at 520 nm. One unit of tannin acyl kinase (67 kDa), fumarase (60 kDa), lactic dehydrogenase (36.5 kDa) hydrolase activity (U) was defined as the amount of enzyme and triosephosphate isomerase (31.5 kDa) were used as molecular necessary to produce 1 μmol of gallic acid per min under the assay mass markers. conditions. For SSF, the activity was expressed as U/mg of solid substrate. 2.7. Influence of temperature and pH on enzyme activity 2.4. Protein quantification and carbohydrate content The enzymatic reaction was conducted at different temperatures (30–80°C).