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
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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
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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)