Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614

AMB Express, Oct 2016

Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn2+, Fe2+, Fe3+ and Mn2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase.

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Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614

Chaitanyakumar and Anbalagan AMB Expr Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614 Amballa Chaitanyakumar 0 M. Anbalagan 0 0 School of Bio‐Sciences and Technology, VIT University , Vellore 632014 , India Enzymes find their applications in various industries, due to their error free conversion of substrate into product. Tannase is an enzyme used by various industries for degradation of tannin. Biochemical characterization of a specific enzyme from one organism to other is one of the ways to search for enzymes with better traits for industrial applications. Here, tannase encoding gene from Staphylococcus lugdunensis was cloned and suitability of the enzyme in various conditions was analysed to find its application in various industry. The recombinant protein was expressed with 6× His tag and purified using nickel affinity beads. The enzyme was purified up to homogeneity, with approximate molecular weight of 66 kDa. Purified tannase exhibited specific activity of about 716 U/mg. Optimum enzyme activity was found to be 40 °C at pH 7.0. Biochemical characterization revealed; metal ions such as Zn2+, Fe2+, Fe3+ and Mn2+ inhibited tannase activity, and SDS at lower concentration, increased tannase activity. Non polar organic solvents increased the tannase activity and polar solvents inhibited the tannase activity. Tannase immobilization studies show protection of the enzyme under wide range of pH and temperature. Also in this study we report a method for recovery and repeated use of the tannase. Tannase; S; lugdunensis; Overexpression; E; coli - Introduction Enzyme catalysed reactions are more preferred over chemical catalyst due to its several advantages; hence enzymes find a wide variety of applications in various industries (Gurung et  al. 2013; Cherry and Fidantsef 2003). Microbes serve as a source for numerous enzymes with wide industrial applications (Demain 2000). Limitations in the quantity of enzyme produced by an organism compels, cloning and over expression of microbial enzymes regularly. Moreover, cloning of genes encoding enzymes of industrial importance is an essential step for engineering of enzymes for better traits. Comparison of an enzyme activity across different species (Taylor et  al. 2002), screening of enzymes with random mutations (Cherry and Fidantsef 2003) and introduction of specific mutations in the enzymes (Neylon 2004; Cherry and Fidantsef 2003) are some of the methods to obtain enzymes for industrial use with desirable traits for various industrial applications. Tannins are polyphenolic compounds produced by plants in order to protect themselves from invading microorganisms and herbivores (Buzzini et  al. 2008). Tannin causes indigestion in herbivores when ingested; sometimes leading to death when ingested in more quantities (Butler 1992). Microbes produce tannase enzyme as a strategy to protect itself from tannin. Tannase enzyme (Tannin acyl hydrolase EC 3.1.1.20) hydrolyzes ester bonds in tannin to produce glucose and gallic acid. Tannase is widely used in industries such as food, chemical, pharmaceuticals, breweries, tannery effluent treatment and production of animal feed (Aguilar et  al. 2007). Tannase is also widely used for the production of gallic acid, which is a key intermediate required for the synthesis of an antibiotic drug, trimethoprim and used to produce propyl gallate, which is mainly used as an antioxidant in fats, oils and beverages (Miura et al. 2013). Gallic © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. acid is also used in the fabrication of semiconductors, dyes and in photographic revelation (Chávez-González et  al. 2012). Thus tannase finds its application in several industries. Given its wide range of applications, there is a need for large scale production of this enzyme and more studies are required for production of good quality and quantity of tannase. Given the little amount of tannase produced by the micro-organisms, cloning and over expression of tannase gene is the feasible process to decrease production cost of the enzyme. Hatamoto et al. (1996) reported first cloning and expression of tannase from Aspergillus oryzae. Fungal tannase gene heterologously expressed in Saccharomyces cerevisiae and Pichia pastoris produced high amounts of recombinant tannase. Fungal tannase is made up of more than one subunit (Yao et  al. 2014), which makes it difficult for over expression and purification. Compared to fungal tannase, bacterial tannase is made up of single subunit (Ren et al. 2013), which makes, cloning over expression and purificat (...truncated)


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Amballa Chaitanyakumar, M. Anbalagan. Expression, purification and immobilization of tannase from Staphylococcus lugdunensis MTCC 3614, AMB Express, 2016, pp. 89, Volume 6, Issue 1, DOI: 10.1186/s13568-016-0261-5