Biochemical Engineering Journal 77 (2013) 161–170 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal jou rnal h om epage: www.elsevier.com/locate/bej Regular article Structural characterization of thermostable, solvent tolerant, cytosafe tannase from Bacillus subtilis PAB2 Arijit Jana, Chiranjit Maity, Suman K. Halder, Arpan Das, Bikash R. Pati, Keshab C. Mondal, Pradeep K. Das Mohapatra ∗ Department of Microbiology, Vidyasagar University, Midnapore 721102, West Bengal, India a r t i c l e i n f o Article history: Received 5 February 2013 Received in revised form 4 May 2013 Accepted 4 June 2013 Available online xxx Keywords: Tannase Bacillus subtilis PAB2 Solid-state fermentation Optimization Downstream processing Kinetic parameters a b s t r a c t Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K m = 0.445 mM, V max = 125.8 mM/mg/min and K cat = 2.88 min –1 ). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 ◦ C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 ◦ C temperature. It exhib- ited half-life (t 1/2 ) of 4.5 h at 60 ◦ C. The enzyme comprised a typical secondary structure containing -helix (9.3%), -pleated sheet (33.6%) and -turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 ◦ C and showed Q 10 , G d and S d values of 2.08, 99.37 KJ/mol and 252.38 J mol -1 K -1 , respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Microorganisms along with their enzymes play an important role in improving several industrial processes. The global mar- ket has a big need for industrial enzymes that is estimated to be worth about 1.6 billion $US, split among food enzymes (29%), feed enzymes (15%) and general technical enzymes (56%) [1]. Produc- tion of feed-grade enzymes such as amylase, cellulase, tannase and phytases by solid-state fermentation (SSF) and their applications in the feed have been examined in many studies [2]. But, the major obstacle against the comprehensive application of these enzymes in industry is their high cost of production as 30–40% of the production cost is accounted for growth substrate [3]. Among the feed enzymes, tannase has attracted much more attention due to its multifaceted potential applications in bever- age, food processing and pharmaceutical industries. The enzyme tannase (EC 3.1.1.20), is an extracellular/intracellular and inducible bio-molecule that increases the bioavailability of nutrients by the hydrolysis of the phenolic (tannin), a potent anti-nutritional fac- tor in plant materials [4]. Tannase is used in wine-making, beer ∗ Corresponding author. Tel.: +91 3222 276554x477; fax: +91 3222 275329. E-mail addresses: arijitjana.mic@rediffmail.com (A. Jana), pkdmvu@gmail.com (P.K. Das Mohapatra). chillproofing, instant tea production and in the pre-treatment of animal feed [5,6]. In addition, it is used as a sensitive analytical probe for detecting cancerous cells [7]. Gallic acid, the prod- uct of tannin hydrolysis, also finds many applications including dye-making, pharmaceutical, leather and chemical industries [5]. However, due to its high production cost it is currently used in a few circumstances and effort should be taken to reduce production cost. Therefore, the use of tannin-rich agro-industrial by products as cheap and readily available substrates, can offer an alternative way of cost effective enzyme production and waste management that can couple with the production of valuable products. Apart from these, there is a substantial gap that still remains in our understanding of catalytic functions and biochemical information about this novel enzyme. Physico-chemical charac- terization of fungal tannase has been extensively studied [8], but information about bacterial tannase is very sparse. Recently, Banerjee et al. [9] have reported the physico-chemical charac- terization, functional motif and evolutionary relationship among fungal and bacterial tannase. It has been widely accepted that the catalytic functions of enzymes are related to protein structure . However, information concerning the tannase structure is scanty. So far, little is known about the tanninolytic machinery of bacteria for tannase production. In this study, an attempt had been made for production of tan- nase in solid state fermentation using tamarind seed as substrate 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.06.002