A novel low molecular weight acido-thermophilic tannase from Enterobacter cloacae MTCC 9125 Vikas Beniwal a,b , Anil Kumar a , Gunjan Goel b , Vinod Chhokar a,n a Department of Bio and NanoTechnology, Guru Jambheshwar University of Science & Technology, Hisar-125001, Haryana, India b Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala-133203, Haryana, India article info Article history: Received 7 February 2013 Received in revised form 3 March 2013 Accepted 7 March 2013 Available online 21 March 2013 Keywords: Enterobacter cloacae Tannase Tannic acid Thermophilic Ultrafiltration abstract Thermophilic tannase from Enterobacter cloacae MTCC 9125 was purified using two-step purification strategy comprising of ultrafiltration and ion-exchange chromatography. A purification fold of 8.47 with 33.1% yield was obtained. The apparent molecular mass of tannase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 31 71 kDa. The temperature of 50 1C and pH 5.5 were found to be optimum for tannase activity. Tannase retained almost 50% activity after 2 and 4 h of incubation at its optimum pH (pH 5.5) and temperature (50 1C), respectively. K m of the enzyme for tannic acid was found to be 0.00337 M with V max 3.401 U/ml. Effects of several metal salts, chelators, surfactants, and typical enzyme inhibitors on tannase activity were evaluated. Inhibition by n-bromosuccinic acid and phenylmethylsulfonyl fluoride (PMSF) indicated that tryptophan and serine or cysteine residues play an important role in maintaining the active conformation of the enzyme. Among the divalent cations, Mg 2 þ , Zn 2 þ and Mn 2 þ were found to be activators of the enzyme whereas Fe 2 þ , Ba 2 þ and Cu 2 þ acted as inhibitors. Surfactant such as Tween 20, Tween 80, Triton X-100 and SDS resulted in considerable loss of enzyme activity. & 2013 Elsevier Ltd. All rights reserved. 1. Introduction Microbial tannase (tannin acyl hydrolase, E.C 3.1.1.20) is an inducible enzyme responsible for the breakdown of ester linkages in tannins and gallic acid esters to produce glucose and gallic acid (Hamdy, 2008). Gallic acid is mainly used in food, cosmetic, hair products, adhesives, and lubricant industries and also as a synthetic intermediate for the production of pyrogallol (Yu and Li, 2006). Gallic acid is extensively used as an ingredient of developer in photography and printing inks. It also serves as a precursor for the commercial production of an anti-microbial drug-trimethoprim, a food preservative-propylgallate and some dyestuffs. Gallic acid possesses wide range of biological activities, such as antioxidant, antibacterial, antiviral, analgesic etc. As an antioxidant, gallic acid acts as an anti-apoptotic agent and helps to protect human cells against oxidative damage. Gallic acid is also found to show cytotoxic activity against cancer cells, without affecting normal cells (Bajpai and Patil, 2008; Beena et al., 2011). Besides gallic acid production, the enzyme is extensively used in the preparation of instant tea, wine, beer, coffee-flavored soft drinks (Belmares et al., 2004; Boadi and Neufeld, 2001) and also as an additive for detannification of food (Mohapatra et al., 2009) and feed (Goel et al., 2005). A potential use of tannase is in the treatment of hard and acidic industrial effluent containing polyphenolic compounds such as tannic acids and as an analytical probe for determining the structures of naturally occurring gallic acid esters (Seth and Chand, 2000). Moreover, it is incorporated into the manufacture of high grade leather (Barthomeuf et al., 1994) and may contribute to plant cell wall degradation by cleaving some of the cross-links existing between cell wall polymers (Battestin and Macedo, 2007; Hamdy, 2008). Microorganisms are the main source for industrial enzymes due to their biochemical diversities, ease of cultivation and amenability to genetic modifications. Bacteria, yeast and filamen- tous fungi are known tannase producers. Most of the organisms capable of degrading tannins isolated till date are either anaerobic or facultative anaerobic bacteria from the alimentary canal of ruminating animals or fungal strains associated with the degrada- tion of wood and forest litter. A major problem in the utilization of fungal strains for industrial applications is that degradation by fungi is relatively slow. It is also difficult to manipulate fungal strains genetically because of their complexity (Beniwal et al., 2010). Although several tannin degrading anaerobic bacteria have been isolated however, their application at industrial scale is limited due to higher cost of maintenance at production level. Keeping these in view, the present investigation reports the purification and Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bab Biocatalysis and Agricultural Biotechnology 1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcab.2013.03.002 n Corresponding author. Tel.: þ91 1662 263 355; fax: þ91 1662 276 240. E-mail addresses: vikashbeniwal@yahoo.com (V. Beniwal), vinodchhokar@yahoo.com (V. Chhokar). Biocatalysis and Agricultural Biotechnology 2 (2013) 132–137