Abstract
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.
Highlights
Enzyme catalysed reactions are more preferred over chemical catalyst due to its several advantages; enzymes find a wide variety of applications in various industries (Gurung et al 2013; Cherry and Fidantsef 2003)
In order to characterize tannase from S. lugdunensis, tannase encoding gene from S. lugdunensis was PCR amplified from its genomic DNA using specific primers
Tannase gene ORF was cloned into BamHI and XhoI site of pET 28a vector multiple cloning site
Summary
Enzyme catalysed reactions are more preferred over chemical catalyst due to its several advantages; 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. Cloning of genes encoding enzymes of industrial importance is an essential step for engineering of enzymes for better traits. Tannins are polyphenolic compounds produced by plants in order to protect themselves from invading microorganisms and herbivores (Buzzini et al 2008). 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
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