d(GC) - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 6 (2) 145-165 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)
not inhibited by Ca ++ , Co ++ , Mg ++ , Mn ++ , Ni ++ even
at 100 mM.
Effect of Denaturants and Surfactants: The
effects of chemical substances on the activity of
an enzyme are often precise and specific.
Surfactants are reported to play key roles in the
catalytic activity of the enzymes. Urea acts as
denaturing agent at about 6 - 8 M (23), by
breaking all hydrogen bonds present in the
protein structure. Although urea is known to inhibit
enzyme activity, it was reported to enhance
maximum tannase activity at a concentration of
1.5 M (109).
Tannase activity was reported at various
concentrations (0.1 - 2.0% w/v) of sodium lauryl
sulphate (SLS) that gradually decreased with
increasing percentage of SLS (23). This inhibition
may be the result of the reduction in the
hydrophobic interactions that play a crucial role
in holding together the protein tertiary structure
and the direct interactions with the protein
molecules. Tween 80 is predominantly
composed of oleic acid (70%) and Tween 20 of
lauric acid (52%) so due to the predominance of
oleic acid and lauric acid, they cause a decrease
in tannase activity. Triton X-100 also caused a
decrease in tannase activity at concentrations of
0.5 and 1.0% (v/v) as reported by Battestin and
Macedo (60). In a similar report, Kar et al. (109),
Tween 60 at 0.05-1.0% (v/v) and SLS, at 0.05-
0.7%, caused inhibition of tannase activity. The
extent of stimulation and inhibition by surfactants
varies for the different enzymes, needs to be
studied thoroughly.
Effect of Chelators and Inhibitors: The
chelators viz., ethylene diamine tetra acetic acid
(EDTA) disodium salt and 1, 10-o-phenanthrolein
at a concentration of 1.0 mM (60) inhibited the
tannase activity. Also the effect of EDTA (1 to 10
mM) on tannase activity was studied and a
concentration of 5 mM was reported completely
inhibitory for tannase activity (23). Tannase from
A. niger and A. oryzae was also inactivated by
157
EDTA, whereas no inhibition was observed in the
case of the tannase from A. flavus (109). The
decreased enzyme activity reported in the
presence of EDTA, could be due to its influence
on the interfacial area between the substrate and
enzyme.
In industrial enzymology, the main
importance of inhibitors is that they reduce the
efficiency of enzyme reaction by altering the
active sites. Studies on P. variable tannase
showed that the enzyme was inhibited by PMSF
and b-mercaptomethanol. Moreover, Nethylmaleimide
showed strong inhibition while
1,10-o-phenanthroline was a mild inhibitor (99).
Similarly R. oryzae tannase was inhibited by
DMSO, b-mercaptoethanol and 1, 10-ophenanthrolien
(109) and A. niger LCF 8 tannase
was inactivated by b-mercaptoethanol (85).
Inhibition studies primarily provide an
insight into the nature of the enzyme, its cofactor
requirements and the nature of the active
enzyme. Tannase activity was found to be
inhibited by sodium bisulphite, iodoacetamide, 2mercaptoethanol,
4-aminobenzoic acid, sodium
azide, n-bromosuccinimide and cysteine at a
concentration of 1 mM. When added to the
reaction medium, cysteine inhibited the tannase
activity of Paecilomyces variotii also the inhibition
of tannase activity by cysteine and 2mercaptoethanol
suggests the presence of
sulphur containing amino acids at the active site
of the enzyme (60).
Stability of Tannase in Non-aqueous
Solvents: Organic solvent stability of tannase is
a very important parameter for its synthesis
activity in non-aqueous media. Synthesis of
various gallic acid esters by tannase takes place
only in non-aqueous solvents. P. variable tannase
(99) showed different degrees of stability in
various organic solvents. More than 60% residual
activity in 20% (v/v) of carbon tetrachloride,
heptane, petroleum ether and toluene after 60
min of incubation, reflects its stability, but in the
Overview on production and characterization of tannases