Chapter-2 Literature Review: 2.0 Sources of Tannase Microbial Source

Optimization of Tannase Production from Agro industrial wastes-Kinetics and Modeling
CHAPTER-2
LITERATURE REVIEW
2.0 SOURCES OF TANNASE
Microbial source
A great variety of bacteria, Klebisella pneumonia (Descamps et al., 1983),

Citrobacter freundii (Kumar et al., 1999), Lactobacillus plantarum (Osawa et al.,
2000), Bacillus lichiniformis (Mondal et al., 2000), Lactobacillus sp.ASR-S1(Sabu
et al., 2006) some yeasts Candida sp (Aoki et al.,1976a,b), Debaryomyces hansenii
(Descamps et al.,1983) and Mycotorula japonica (Belmares et al., 2004) are
known as tannase enzyme producers. Mainly fungi are considered as the best
enzyme producers. Filamentous fungi of the Aspergillus and Penicillium genus
have been widely used for tannase production. Fungi like Aspergillus oryzae
(Bradoo et al., 1996) Aspergillus awamori (Beena et al., 2010), Aspergillus
fumigates (Batra and Saxena., 2005), Aspergillus ruber (Kumar et al., 2007),
Penicillium chrysogenum (Bradoo et al., 1996), Penicillium glabrum (Vande
Lagenaat and Pyle., 2005), Trichoderma viride and Trichoderma hamatum
(Bradoo et al., 1996) are reported as good tannase producers.
Plant source
Lekha and Lonsane., (1997) studied the production of tannase and reported
the presence in many tannin-rich plant materials, such as myrobolan (Terminalia
chebula) fruits, divi-divi (Caesalpinia coriaria) pods, dhawa (Anogeissus latifolia)
leaves and the bark of konnam (Cassia fistula), babul (Acacia arabica) and avaram
(Cassia auriculata) trees. Plant tannase is found to be less stable compared to
microbial sources and purification is more cumbersome.
Animal source
Sabu et al., (2006) reported many gastrointestinal bacteria of adopted
domestic and wild animals found to produce tannase and several species of these
bacteria have been isolated from faeces of koalas, goats and cows.
8
2.1 TANNASE SUBSTRATES
Tannins are naturally occurring water-soluble polyphenolic compounds

with varying molecular weight that occur naturally in the plant kingdom. These
phenolic compounds differ from others by its ability to precipitate proteins from
solutions. In the plant kingdom these tannins are found in leaves, fruits, bark and
wood. They occur in many edible fruits and vegetables and are often considered
nutritionally undesirable because they form complexes with protein, starch and
digestive enzymes and cause a reduction in nutritional value of food (Chung et al.,
1998). Tannins are classified into two groups, hydrolysable tannins and condensed
tannins (Bhat et al., 1998). Current and the most accepted classification divides the
tannins into four groups namely gallo tannins, ellagi tannins, condensed tannins
and complex tannins (Fig.2.1.1). Tannins are in fact antimicrobial agents and most
of the microorganisms cannot tolerate its polyphenolic nature. Only a few
microorganisms can degrade tannic acid and utilize it as nutrient (Lekha and
Lonsane., 1997).
(a) Gallotannins are the simplest tannins that exist and are formed by units of
galloyl or di-galloyl esterified to a core of glucose or other polyhydroxy
alcohol. Upon hydrolysis, gallotannin such as Chinese gallotannin (Rhus
semilata) and sumac tannin (Rhus coriaria) yield glucose and phenolic
acids mainly gallic acid.
(b) Ellagitannins are esters of hexahydrodiphenic acid (HHDP) and during its
hydrolysis, the HHDP group dehydrates and lactonize spontaneously to
form ellagic acid. On hydrolysis, ellagitannins like myrobolan (Terminalia
chebula) tannin and divi-divi (Caesalpinia coriaria) tannin yield glucose
and ellagic acid together with gallic acid and frequently other acids
structurally related to gallic acid (Haslam et al., 1961).
(c) Condensed tannins are oligomeric and polymeric proanthocyanidins
containing flavan-3-ol (catechin) or flavan-3,4-diol linked by C–C– bonds.
In contrast to the hydrolysable tannins, they do not contain sugar residues
(Goodwin and Mercer., 1983) and are less susceptible to microbial and
chemical attack. Examples of condensed tannins include wattle (Acacia
9
mollisima) tannin and quebracho (Schinopsis lorentzii) tannin (Lewis and

Starkey., 1969).
(d) The basic structure of complex tannins consists of a gallotannin or
ellagitannin unit and one of catechin (Belmares et al., 2004; Aguilera-
Carbo et al., 2008).
Fig.2.1.1 Classification of Tannins ( Khanbabaee and Van Ree., 2001)

2.1.1 Sources of Tannin
Tannins are natural polyphenolic compounds present in vascular plants.
They are characterized by their ability to form strong complexes with different
minerals and macromolecules, such as proteins, cellulose, starch, among others.
Due to their strong ability to bind with proteins, they have been used in tanning for
thousands of years (Aguilar et al., 2007). Application of agro industrial residues as
substrates is certainly economical and it also reduces environmental pollution.
Tannin are present in several naturally occurring agricultural wastes such as
redgram husk, greengram husk, blackgram husk, tamarind seed powder, tea dust,
rice bran and groundnut shell could be used in one or the other industrial
bioprocess for the production of value added products through Submerged
fermentation. Tannin content of the materials are listed in Table 2.1.
10
Table 2.1 Sources of Tannin

Material Tannin content Reference
(mg/g)
Redgram husk 2.601 Paranthaman et al., (2009)
Greengram husk 0.892 Prakasam et al., ( 2010)
Blackgram husk 0.910 Arulnathan et al., (2013)
Tamarind seed powder 1.202 Siddig et al., (2006)
Tea dust 0.102 Gowdhaman et al., (2012)
Rice bran 0.096 Paranthaman et al., (2009)
Groundnut shell 0.172 Paranthaman et al., (2009)
2.2 BIODEGRADATION OF TANNINS

Biodegradation by certain microorganism and enzymes is one of the most
efficient ways to degrade large tannin molecules into small molecules with bio-
activities of high value. The ability of microorganism to assimilate tannin differs
among yeast, bacteria and fungi. Yeast, while acting effectively against
gallotannins, loses it degradation ability against high molecular compounds.
Bacteria have the ability to degrade gallotannins as well as ellagitannins. Fungi can
efficiently degrade different types of tannins (Bhat et al., 1998).
Some of the enzymes involved in degradation of gallotannins are tannase
and gallic acid decarboxilase. Tannase is perhaps the most studied enzyme so far in
the biodegradation of tannins. It has act on ester and depside bonds of gallotannin
and may be microbial, plant, or animal in origin, of which microorganisms are the
most important source (Aguilar et al., 2007). Tannase acts on gallotannins,
ellagitannins, and complex tannins by breaking only ester bonds without affecting
the carbon–carbon bonds and hence does not affect the condensed tannins (Haslam
and Stangroom., 1966).
Gallic acid decarboxylase (E.C. 4.1.1.59) can catalyze the decarboxylation
of gallic acid to pyrogallol. This enzyme is very unstable because of its high
sensitivity to oxygen, and thus, it is difficult to isolate and purify (Zeida et al.,
1998).Some bacteria, such as selenomonas gallolyticus and Escherichia coli,
decarboxylate gallic acid to pyrogallol, but this compound is not further
11
transformed. The reason is unclear but is likely to be less toxic than gallic acid or
that its production is thermodynamically more favorable and is possibly linked to
the generation of energy by pumping protons (Mingshu et al., 2006).
In the case of ellagitannin biodegradation, the release of ellagic acid has
been attributed to a new enzyme (ellagitannin acyl hydrolase). However, it is
necessary to perform a study to demonstrate the catalytic difference between tannin
acyl hydrolase and ellagitannin acyl hydrolase and to understand the
biodegradation processes of gallotannins and ellagitannins (Aguilera-Carbo et al.,
2008). On the other hand, the study of the degradation of condensed tannins and
complex is much more difficult due to their complicated structures. Therefore,
there is little progress in understanding the mechanisms of degradation of these
compounds. The biodegradation metabolic pathway of gallotannins is presented in
Fig. 2.2.1
Fig.2.2.1 Gallotannin enzymatic biodegradation pathways (Mingshu et al., 2006)
12
2.2.1 Bacterial Degradation

Certain bacteria of the genera Bacillus, Staphylococcus, Klebsiella, and
Lactobacillus (Deschamps et al., 1980; Ayed and Hamdi., 2002) have the ability to
degrade tannins. Gallic acid monomers are used as a substrate after a break of
simple aliphatic acids. Gallic acid is converted to pyrogallol by the enzyme gallate
decarboxylase or gallate carboxylyase. The anaerobic decomposition of gallic acid
and hydrolyzable tannin monomer occurs by different mechanisms. The first step
is decarboxylation of gallic acid to form pyrogallol, which is converted to
phloroglucinol by pyrogallol phloroglucinol isomerase and to
dihydrophloroglucinol by phloroglucinol reductase. Later, dihydrophloroglucinol
is converted to 3-hydroxy-5- oxohexanoate (HOHN) by dihydrophloroglucinol
hydrolase. The HOHN is degraded by different pathways by rumen
microorganisms. Under anaerobic conditions, HOHN is converted to 3,5-
docosahexaenoate (triacetate) by the HOHN dehydrogenase and finally to three
molecules of acetyl-CoA by the sequential enzymatic action of triacetyl-CoA
transferase, triacetate ß-ketothiolase, acetoacetyl-CoA ß-ketothiolase,
phosphotransacetylase, and acetate kinase (Brune and Schink., 1992). HOHN can
be converted to acetate and butyrate, too, in rumen ecosystem. HOHN-CoA is
derived from the enzymatic action of HOHN-CoA transferase and is converted to
acetate and butyrate by the rumen bacterial by the sequential action of ß-
hydroxybutyryl-CoA dehydrogenase, butyryl-CoA dehydrogenase, acetyl-CoA
acetyltransferase, enoyl Co-A hydratase, phosphate acetyl transferase, and acetate
kinase (Brune and Schink., 1992; Nelson et al., 1995)
2.2.2 Fungal Degradation
The degradation of hydrolyzable tannins particularly gallotannins is best
known in fungal systems. The oxidative degradation of hydrolyzable tannins has
been studied in Aspergillus sp. (Cruz-Hernández et al., 2009; Belmares et al.,
2003). While routes of degradation of gallic acid have been determined, the
degradation pathway of hexahydroxydiphenyl and proanthocyanidins is still not
well understood.
13
In Aspergillus niger, oxygenase converts gallic acid to unstable

intermediate of tricarboxylic acid. This is finally decarboxylated by an oxidative
decarboxylase to form cis-aconitic acid, which enters the citric acid cycle.
However, in Aspergillus flavus, gallic acid is degraded to oxaloacetic acid and
finally to pyruvic acid (William et al., 1986). Pyrogallol, the decarboxylated
derivative from gallic acid, is also oxidized to cis-aconitic acid by the same
mechanism.
The metabolism of hydrolyzable tannins, especially tannic acid has
received considerable interest (Ajay Kumar et al., 1999). It is well known that the
degradation of tannic acid by the action of the enzyme tannase releases gallic acid
and glucose. Attempts have been made to characterize the intermediate metabolites
of this action and found pyrogallol in addition to glucose and gallic acid. Ajay-
Kumar et al., (1999) reported the route of degradation of tannic acid by Citrobacter
freundii and observed that glucose formed by tannin hydrolysis enters glycolysis
and subsequently enters the tricarboxylic acid cycle. Gallic acid is converted to
pyrogallol by gallic acid decarboxylase. The presence of pyrogallol and resorcinol
has been observed in vitro studies with rumen systems (Singh et al., 2001).
Pyrogallol is converted to hydroxymuconic acid by pyrogallol dioxygenase
dioxygenase, which is later converted to pyruvate and it is further metabolized
through tricarboxylic acid cycle.
2.3 PHYSICOCHEMICAL PROPERTIES OF MICROBIAL TANNASE
Physicochemical properties of tannase, resumed in Table 2.3.1, are one of
the most discussed topics. Tannase properties depend on the source and culture
conditions; for example, all characterized tannases from yeast and fungi are
glycoproteins, but bacterial tannases seem not to present such post-translational
modifications. In addition, it has been found significant differences on
glycosylation between tannases produced in different culture systems by the same
microorganism (Renovato et al., 2011). Tannase glycosylations consists of neutral
sugars in the range from 5.4% to 64%, and it may be related to the resistance of the
TAH to be precipitated by tannins, as most of the proteins (Aoki et al., 1976b;
Beverini and Metche., 1990; Zhong et al., 2004; Kasieczka-Burnecka et al., 2007).
14
2.3.1 Optimum temperature and temperature stability
The temperature dependence of many enzyme-catalyzed reactions can be

described by the Arrhenius equation. An increase in the temperature increases the
rate of reaction, since the atoms in the enzyme molecule have greater energies and
a greater tendency to move. However, the temperature is limited to the usual
biological range. As the temperature rises, denaturation processes progressively
destroy the activity of enzyme molecules. This is due to the unfolding of the
protein chain after the breakage of weak (hydrogen) bonds, so that the overall
reaction velocity drops. For many proteins, denaturation begins to occur at 45°C to
50°C. Some enzymes are very resistant to denaturation by high temperature,
especially the enzymes isolated from thermophilic organisms found in certain hot
environments (Rajiv Dutta., 2008).
Batra and Saxena., (2005) reported that the temperature range of the
tannase production as 30°C–70°C and the optimum temperature as 60°C for
A.flavus, A.fumigatus, A.versicolor and P. variable, whereas A. caespitosum, P.
charlesii, P. crustosum and as 40°C for P. restrictum. Tannase obtained from A.
versicolor was more stable in a broad temperature range of 30°C –80°C.
Hina Iqbal et al., (2012) studied the effect of temperature on tannase
enzyme production using T.harzianum in the temperature range from 25°C to 60°C
and the optimum temperature was found to be 40°C and was stable at 40ºC
retaining about 71% of original activity for 2 hours. Costa et al., (2008) obtained
similar results for tannase enzyme production using A.tamari in submerged culture
fermentation and the optimum temperature was found to be 35ºC and was stable at
40ºC for 2 hours.
Banerjee et al., (2007) investigated the possible use of wheat bran as
substrate for production of tannase and reported a maximum tannase activity of
8.16 U/g at 30°C by Aspergillus aculeatus DBF9. Kumar et al., (1999) used a
mineral medium with a maximum tannic acid concentration of 5% (w/v) in the
production of tannase using Citrobacter freundii and reported a maximum tannase
activity of 12.16 U/ml at 30°C. Selwal et al., (2010) studied the optimization of
process parameters for tannase production using Pseudomonas aeruginosa IIIB
15
8914 and reported a maximum tannase activity of 13.16 U/ml at 37°C. Bradoo et
al., (1997) reported that the production of tannase using Aspergillus japonicas was
found to be 33.06 U/ml as maximum tannase activity with 0.2% glucose and 2%
tannic acid in Czapek- Dox's Minimal Medium at pH 6.6.
Renovato et al., (2011) studied the production of tannase using A.niger
under solid state fermentation and submerged fermentation. The optimum
temperature and pH range were found to be from 50°C to 60°C and 5–8
respectively in submerged fermentation. The optimum temperature and pH were
found to be 60°C and 6 respectively in solid state fermentation.
2.3.2 Optimum pH and Stability
All proteins are constructed from various amino acids. These biochemical
units possess basic, neutral or acidic groups. Consequently, the intact enzyme may
contain both positively or negatively charged groups at any given pH. Such
ionizable groups are often apparently part of the active site since acid- and base-
type catalytic action has been linked closely to several enzyme mechanisms. For
the appropriate acid or base catalysis to be possible, the ionizable groups in the
active site must often each possess a particular charge; i.e., the catalytically active
enzyme exists in only one particular ionization state. Thus, the catalytically active
enzyme may be a large or small fraction of the total enzyme present, depending
upon the pH (Bailey and Ollis., 1944).
Aoki et al., (1976 a) studied the production of tannase from Candida sp.
K-1 and showed an optimum activity at a pH value of 6.0. The investigation also
revealed that the enzyme was stable over a wide pH range of 3.5 to 7.5. Rajakumar
and Nandy.,(1983) reported the isolation and purification of tannase from
Penicillium chrysogenum and showed broad pH dependence in the range from 5 to
6, with the optimum enzyme activity and was stable in the pH range of 4.0 to 6.5 at
16°C.
Iibuchi et al., (1968) reported the effect of pH on tannase production using
A. oryzae and the tannase was stable in the pH range of 4.5 to 6.0 for 25 hours with
an optimum pH of 5.5. Anwar et al., (2009) studied the production and
characterization of tannase using A.niger isolated from cacao pod in a solid state
16
fermentation with wheat flour as a substrate and obtained a maximum tannase

activity of 10.63 U/ml at pH 6.0. Niehaus and Gross., (1997) studied the tannase
production using penduculate oak as a substrate and the optimum pH was found to
be 5.0.
Mahendran et al., (2006) studied the purification and characterization of
tannase using P. variotii and the enzyme was stable at a pH from 5 to 7 and the
optimum pH was found to be pH 6. Mahapatra et al., (2005) reported the
production of tannase using A.awamori nakazawa and exhibited optimum activity
at 35°C and at a pH of 5.
2.3.3 Molecular Weight of Tannase
The molecular weight of tannases was found to be in the range of 50 kDa –
320 kDa (Iwamoto et al., 2008; Boer et al., 2009). It has been reported that
tannases consist of two or more subunits. Hatamoto et al., (1996) concluded that
native tannase of Aspergillus oryzae consists of four pairs of two types of subunits
(30 kDa and 34 kDa, respectively) linked together by disulfide bonds, forming a
hetero-octamer of 300 kDa. Mahendran et al., (2006) reported the molecular
weight of tannase using Pacilomyces variotii was reported to be 149.8 kDa through
native PAGE analysis with a monomeric unit of molecular mass 45kDa by SDS-
PAGE analysis. Renovato et al., (2011) studied the SDS-PAGE analysis of both
SSF and SmF purified tannases showed a single band with a molecular weight of
102 kDa and 105 kDa, respectively.
2.3.4 Kinetic Parameters of Tannase
Substrate affinity for tannase from several fungi has been found to be
different. The Km values were 0.28, 0.95, 1.05, and 0.048 for tannase from A.
niger, Cryphonectria parasitica, Verticillium sp., and Penicillium chrysogenum,
respectively, when tannic acid was used as substrate and reaction was carried out at
30°C and pH 5.0–6.0 (Bhardwaj et al., 2003; Farías et al., 1994; Kasieczka-
Burnecka et al., 2007; Rajakumar and Nandy., 1983). However, caution should be
taken when comparing these values due to the varying quality of the substrates
utilized and the different methods utilized to quantify the reaction product. Anwar
et al., (2009) reported the kinetics analysis that showed that Km and Vmax value of
17
tannic acid was 0.401 mM and 10.804 U/ml respectively and Gallotannin yielded
Km and Vmax values of 6.611 mM and 12.406 U/ml respectively. Battestin and
Macedo., (2007) reported the Km and Vmax values were found to be 0.61 μmol and
0.55 U/ml with wheat bran and coffee husk as used as the substrate.
2.3.5 Effect of Carbon Sources
Selwal et al., (2011) studied the effect of carbon sources on production of
tannase using tannic acid, dextrose, glucose, glycerol, maltose, manitol, lactose and
sucrose. The strain P.atramentosum KM produced maximum tannase of 29.4 U/ml
with amla leaves and 31.1 U/ml with keekar leaves as substrates when
supplemented with 0.2% (w/v) maltose. Lokeswari et al., (2007) studied the effect
of different glucose concentrations on tannase production using Aspergillus niger.
A maximum tannase activity of 21.42 U/ml with 0.5% (w/v) glucose concentration
was reported.
Deepanjali Lal et al., (2012) studied production of tannase using Aspergillus
niger with different carbon sources namely tannic acid, mannose, galactose,
glycerol and ribose. Tannic acid (1% w/v) was the most suitable carbon source for
tannase production.
2.3.6 Effect of Additives
The effect of additives on tannase activity has been studied by several
authors. Kar et al., (2003) studied the effect of metal ions on a Rhizopus oryzae
tannase. They found that 1 mM of Mg+2 or Hg+ activated tannase activity, whereas
that Ba+2, Ca+2, Zn+2, Hg+2, and Ag+ inhibited tannase activity, and Fe+3 and Co+2
completely inhibited tannase activity. On the other hand, the tannase from A. niger
GH1 was highly inhibited by Fe+3, whereas Cu+2 and Zn+2 had only a mild
inhibitory effect, and Co+2 enhanced the enzyme activity (Mata-Gómez et al.
2009). Furthermore, Mg+2, Mn+2, Ca+2, Na+ , and K+ stimulated the activity of
Aspergillus awamori tannase, while Cu+2, Fe+3, and Co+2 acted as inhibitors of the
enzyme (Chhokar et al., 2010a).
18
Table 2.3.1 Physicochemical Properties of Tannase
Microorganism Optimum pH, Other properties Reference

pH and Temperature
Optimum Stability
Temperature
Aspergillus pH 6.0, 60°C Stable at pH Km = 0.20 mM, Sharma
niger van 3.0 –8.0, Vmax = 5.0 μmol et al., (1999)
Tieghem temperature min−1 mg−1 protein
30 °C – 60°C
Aspergillus pH 6.0, 40°C Enzyme was Enzyme activity Sabu et al.,
niger ATTC active at pH was inhibited by (2005)
16620 4.0-8.0, Zn2+, Mn2+, Cu2+,
30°C – 40°C Ca2+, Mg2+ and
Fe2+, and enhanced
by K+.
Aspergillus pH 6.0, 60 °C Stable at pH The enzyme was Mata Gomez
niger GH1 3.0–6.0, highly inhibited by et al., (2009)
stable at Fe3+, whereas Cu2+
temperatures and Zn2+ had only a
lower than mild inhibitory
50°C effect. Co2+
enhanced the
enzyme activity.
A.niger LCF 8 pH 6.0, 35 °C Stable at pH Molecular weight- Barthomeuf et
3.5 –8.0, 186000 kDa al., (1994)
temperature Inhibitor- CuSo4
4 °C – 45°C (68%),
ZnCl2 (39%)
Candida sp. pH 5.5, 50 °C Stable at pH Molecular weight- Aoki et
K 16 3.5 –7.5, 250000 kDa al.,(1976)
temperature Protein content-
4 0°C 35%
Aspergillus pH 5.0, 60°C Stable at pH Both intracellular Banerjee

aculeatus for 4.0–6.0, and extracellular et al., (2007)
DBF 9 intracellular Stable for 1 h enzymes are salt
enzyme, at 50°C tolerant up to 3
50 °C for M NaCl
extracellular
enzyme
Penicillium pH 5.0, 50°C Active at pH Mol.wt- 2 kDa Sharma

variable 3.0–8.0, Km = 32 mM, et al ., (2008)
25–80°C Vmax = 1.1 μmol
min−1 ml−1
19
Pencillium pH 5.0-6.0 Stable at Km = 0.48 x 10-4 M Rajakumar

chrysogenum 30°C -40°C pH 4.0–6.5, and Nandy.,
stable (1983)
temperature
upto 30°C
Bacillus cereus pH 4.5, 40°C Stable at Enzyme is salt Mondal

KBR9 pH 4.5–5.0, tolerant, stable up et al., (2001)
stable to 2 M of NaCl and
temperatures retains 82%
lower than original activity in
30°C 3 M.
Lactobacillus pH 5.0, 30°C Stable at Enzyme activity Rodríguez

plantarum 22°C –37°C was not affected by et al., (2008)
CECT 748T K+, Ca2+, Zn2+,
Tween 80, EDTA
and urea, but
inhibited by Hg2+
Lactobacillus pH 8.0, 40°C Active at The enzymatic Curiel et al.,

plantarum pH 6.0–8.0, activity was (2009)
ATCC 1491 T stable at increased by K+
(recombinant) temperatures and Ca2+, not
lower than significantly
30°C affected or partially
inhibited by EDTA,
Mg2+, Zn2+, Triton-
X-100, Tween-80
Paecilomyces pH 6.0, 40°C Stable at Mol.wt- 45 kDa Mahendran

variotii pH 4.0–8.0, et al., (2006)
30°C –50°C
A.oryzae pH 5.5, Stable at Molecular weight- Iibuchi et

30°C - 40°C pH 3.0–7.5, 200000 kDa al.,(1972)
30°C Inhibitor-
Cu and Zn
20
2.4. STRUCTURE OF TANNASE
Enzyme name : Tannase

Enzyme Commission number: EC 3.1.1.20
Structure : Crystalline structure
Secretion : Extracellular
Reaction : digallate + H2O = 2 gallate
Other name(s) : Tannase S, Tannin acetylhydrolase
Systematic name: Tannin acylhydrolase
Comments: Also hydrolyses ester links in other tannins.
Fig.2.4.1 Structure of Tannase
2.5 PRODUCTION OF TANNASE

Studies on tannase production by bacteria, fungi and yeast have been
carried out under submerged and solid state fermentation conditions. Depending on
the strain and the culture conditions, the enzyme was induced and expressed with
different levels of activity, showing different production patterns. Tannase
synthesis was induced by phenolic compounds such as methyl gallate, gallic acid,
pyrogallol, and tannic acid (Rana and Bhat ., 2005).
2.5.1 Tannase Production in Submerged Fermentation
Submerged fermentation is always preferred for microbial tannase
production because it offers uniform fermentation conditions like substrate
21
concentration, inducer concentration, temperature, pH, dissolved oxygen

concentration, agitation and aeration. Microbial tannase production belongs to
aerobic fermentation and submerged fermentation is a better choice to provide
uniform aeration conditions. Sometimes solid state fermentation methods are
adopted for tannase production since cheaper agricultural residues based media
could be used. The disadvantages of solid state fermentation are difficulties in
scale up, control of process parameters (pH, heat, moisture, nutrient conditions
etc.,) and quality of the product.
Hina Iqbal et al., (2012) studied the production of tannase using
Trichoderma harzianum MTCC 10841 under submerged fermentation with rich
tannin materials like amla (Phyllanthus amblica, bark, leaves and fruit), amaltash
(Cassia fistula, leaves), ber (Zyzipus maurtiana, leaves), Eucalyptus (Eucalyptus
glogus, bark and leaves), jamun (Syzgium cumini, bark and leaves), guava (Psidium
guazava, bark and leaves), keekar (Acacia nilotica, leaves), mango (Magnifera
indica, leaves), mulberry (Morus macroura, leaves), tamarind (Tamarindus indica,
seed) and pomegranate (Punica granatum, rind) as carbon sources. Amla fruit,
tamarind seed, jamun leaves, mulberry leaves and keekar leaves proved to be best
substrates than tannic acid and the optimum pH and temperature for the tannase
enzyme production were found to be 5.5 and 40ºC respectively.
Das Mohapatra et al., (2006) used eight different plant extracts as the
tannin source for tannase production using Bacilus lichiniformis KBR6 under
submerged fermentation and obtained highest activity from the crude extract of
Anacardium occidentale. Darah et al., (2011) reported the production of tannase
by submerged fermentation using Aspergillus niger FETL FT3 with an initial
medium pH of 6.0 at 30ºC, agitation speed of 200 rpm and inoculums size of
6 x 106 spores/ml. Maximum tannase production of 2.81 U/ml was obtained on
the fourth day of cultivation.
Selwal et al., (2010) studied the production of tannase enzyme using
Pseudomonas aeruginosa IIIB 8914 under submerged fermentation with the leaves
of Phylanthus emblica (amla), Acacia nilotica (keekar), Eugenia cuspidate (Jamoa)
and Syzygium cumini (Jamun) as substrates and reported a maximum tannase yield
22
of 13.65 U/ml and 12.90 U/ml were obtained using amla and keekar leaves
respectively.
Wilson Peter et al., (2009) used a bacterial isolate, Citrobacter sp., from
tannery effluent has proved as a potent producer of tannase enzyme. The
production by solid state fermentation of tannase was compared with submerged
fermentation using tamarind seed as sole carbon source. Two times higher tannase
activity was observed in solid state fermentation (90 U) than submerged
fermentation (50 U) at 48 hours with 5 g of substrate.
Murugan et al., (2007) isolated 10 morphological different fungal strains
from a tannery effluent. Selected microorganisms were tested for tannase
production under submerged fermentation in a stirred tank bioreactor and reported
strain A.niger MS101 as the best tannase producer.
Sharma et al., (2007) studied the optimization of tannase production by
statistical techniques using Aspergillus niger in submerged fermentation. The
effect of concentrations of tannic acid, sodium nitrate, agitation rate and incubation
period on tannase production was studied. 5% tannic acid, 0.8% sodium nitrate,
150 rpm agitation and 48 hours were found to be optimum conditions and gave a
maximum tannase activity of 19.7 U/ml.
Beniwal et al., (2010) reported the optimization of culture conditions for
tannase production by Aspergillus awamori MTCC 9299 using the response
surface methodology. Maximum yield of tannase production was obtained at pH of
5.0, incubation temperature of 35°C, agitation speed of 125 rpm and 48 hours of
incubation period. Under the proposed optimized conditions, the tannase
experimental yield of 1.45 U/ml was closely matched the yield predicted by the
statistical model of 1.43 U/ml with R2 value of 0.99.
Kannan et al., (2011) studied the production of tannase by Lactobacillus
plantarum MTCC 1407 under submerged fermentation. Maximum tannase activity
of 5.22 U/ml was obtained at 24 hours using the following medium (g/L): tannic
acid-10: glucose-1; NH4Cl-3; MgSO4.7H2O-2; KH2PO4 - 0.5; K2HPO4 - 0.5;
CaCl2-1.
23
The summary of the production of tannase enzyme using various microorganisms

by submerged fermentation was given in Table.2.5.1
Table.2.5.1 Tannase Production by Submerged Fermentation
Enzyme
Strain Substrate Process conditions Reference
activity
pH -6.6
Temperature -35°C
Bradoo et al.,
A.japonicus Tannic acid Incubation time-72 hrs 33.06 U/ml
(1996)
Substrate conc -5%
Tannic acid conc -3%
Citrobactor Tamarind pH -5.5 50 U Wilson Peter
species seed powder Temperature- 35°C 90 U et al., (2009)
pH -5.5 Lokeswari
A. niger Tannic acid 120 U/ml
Temperature -35°C et al., (2007)
pH -5
A.oryzae 643 Cashew Lokeswari
Temperature- 40°C 32.62 U/ml
(NCIM) husk et al., (2010)
Incubation time -24hrs
pH -5
Pomegra Srivastava et
A. niger Temperature - 37°C 28.72 U/ml
nate rind al., (2009)
Incubation time -72hrs
pH -6
Lokeswari
A.niger Tannic acid Temperature -50°C 65 U/ml
et al., (2006)
Incubation time - 96hrs
Temperature - 35°C
Paranthaman
A.flavus Tannic acid Incubation time - 96hrs 30 U/g/min
et al., (2009)
Tannic acid conc - 3%
pH-5.5
Trichophyton
Temperature -30°C 36.54 Krishnasamy
rubrum Tannic acid
Incubation time – 30hrs U/g/min et al., (2009)
MTCC
Tannic acid conc -2.5%
Chhokar
pH -5.5
A. awamori Tannic acid 1.43U/ml et al.,
Temperature - 30°C
(2010a)
Amla
Ber pH -5
Temperature -35°C 13.65U/ml Manjit
P. aeruginosa Jamun Incubation time -24hrs 12.90U/ml et al., (2010)
Jamoa
Keekar
pH -5
Temperature -30°C Deepanjali
101.428
A.niger Tannic acid Incubation time - Lal et al.,
U/ml
168hrs (2012)
24
2.5.2 Tannase Production in Solid State Fermentation

Sabu et al., (2005) studied the production of tannase under solid-state
fermentation using A. niger ATCC 16620 with palm kernel cake and tamarind seed
powder as the substrate. A maximum enzyme yield of 13.03 IU/g dry substrate
(gds) was obtained at 30°C and 5% tannic acid as additional carbon source after 96
hours of fermentation.
Kumar et al., (2007) investigated tannase production using A. ruber by
solid state fermentation using different tannin rich agro-wastes namely ber leaves
(Zyzyphus mauritiana), jamun leaves (Syzygium cumini), amla leaves
(Phyllanthusemblica) and jawar leaves (Sorghum vulgaris). Jamun leaves were
found to be the best substrate for tannase production and a maximum production of
tannase 30.2 U/ml was recorded at 30.1ºC after 96 hours of incubation.
Bhaskar Reddy and Vandhana Rathore., (2012) studied the production of
gallic acid using tannase enzyme. Tannase enzyme was produced using acacia
pods, redgram husk, sorgum husk and spent tea powder as substrates through solid
state fermentation by the isolate P.purpurogenum BVG 7.The effect of pH and
temperature on tannase production was studied and pH 5.5 at 30 ºC were found to
be optimum conditions for maximum tannase activity and gallic acid production.
Battestin et al., (2007) studied the production of tannase using
Paecilomyces variotti with coffee husk and rice bran as the substrate for solid state
fermentation and evaluated the effects of variables namely temperature (⁰C), tannic
acid (%), residue (%) (coffee husk: wheat bran) and incubation time on the
production of tannase. The optimum conditions for tannase production by
Response Surface Methodology were found to be temperature (29⁰C –34⁰C), tannic
acid (8.5–14%); % residue (coffee husk:wheat bran 50:50) and incubation time of
5 days.
Kulkarni et al., (2012) studied the tannase production using A.oryzae with
mixture of Arjuna (Terminalia arjuna), Babul (Acacia nilotica), Jamun (Syzygium
cumini), Mulberry, Chiku stem barks as substrates by solid state fermentation. The
mixture of Jamun and Babul bark in the ratio of 4:6 was reported to be best
substrate and gave a maximum tannase activity of 116 U/g dry substrate.
25
Aguilar et al., (2001) reported the production of tannase using Aspergillus

niger Aa-20 in solid state fermentation and submerged fermentation with tannic
acid and glucose as carbon sources. The biomass yield in solid state fermentation
was found to be 2 times higher than in submerged fermentation.
Pinto et al., (2001) investigated the tannase activity of 17 wild type and 13
mutant strains of Aspergillus niger from a local culture collection in Brazil
(EMBRAPA / Food Technology stock collection) and selected the potential
tannase producers for maximum tannase production by solid state fermentation.
Paranthaman et al., (2009) did a comparative study on the suitability of
different substrates for maximum tannase production using A.oryzae in solid state
fermentation using various agricultural by-products. Rice bran was reported to be
the best substrate for the tannase enzyme production and gave a maximum tannase
activity of 14.40 U/g/min at 30⁰C, pH 5.5 in 96 hours.
Manjit et al., (2008) studied the production of tannase by solid-state
fermentation using Aspergillus fumigatus MA with different agro forest residues
such as Amla leaves (Phyllanthus emblica), Ber leaves (Zyzyphus mauritiana),
Jamun leaves (Syzygium cumini), Jamoa leaves (Syzygium sp.) and Keekar leaves
(Acacia nilotica) as substrates. The maximum tannase activity of 174.32 U/g was
obtained using Jamun leaves as substrate at 25⁰C, pH 5 in 96 hours of incubation.
Rodrigues et al., (2008) studied the effects of inoculum concentration,
temperature, and carbon sources on tannase production by solid state fermentation
with cashew apple bagasse. The maximum tannase activity of 4.63 U/g of dry
substrate was obtained at 30°C, using 107 spores/g with 1.0% (w/v) sucrose as an
additional carbon source.
Narsi reddy et al., (2011) reported tannase production using red gram husk,
green gram husk, ground nut waste, cotton seed waste, wheat bran, rice bran,
coffee husk, tamarind seed powder, cashew apple bagasse, coconut powder, corn
powder and cyceraritinum as substrates by solid-state fermentation using isolated
A.terreus. A maximum tannase activity of 41.6 U/mg was reported with wheat bran
as a substrate at pH of 3.5 and incubation period of 72 hours.
26
Kannan et al., (2012) studied production of tannase using Lactobacillus

plantarum MTCC1407 in submerged fermentation and solid state fermentation
process. They optimized the nutrients using Plackett–Burman screening and
response surface methodology. Maximum tannase activity of 9.13 U/ml was
observed at 30 hours of fermentation. Solid state fermentation was conducted with
various solid substrates and agricultural residues. Maximum tannase activity of
5.319 U/gds was obtained with coffee husk as substrate.
Boer et al., (2011) studied the large scale production of tannase using
Arxula adeninivorans strains in Fed-Batch fermentation. Transformant strains that
overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter
produce levels of up to 1,642 U / l when grown in glucose medium in shake flasks.
The effect of Fed-Batch fermentation on tannase productivity was found to be
51,900 U / l after 142 hours of fermentation at a dry cell weight of 162 g / l.
Zhong et al., (2004) cloned and expressed the tannase gene using A.oryzae
in the methylotrophic yeast Pichia pastoris and obtained large quantities of
extracellular tannase was found to be 7,000 U/l in a short period of 96 hours
incubation in a SmF Fed-Batch system.
27
The production of tannase using various microorganisms by solid state fermentation

was summarized in Table.2.5.2
Table.2.5.2 Tannase Production by Solid State Fermentation
Process Enzyme
Strain Substrate Reference
Conditions Activity
pH - 5.5
Sugarcane
Temperature -
A.oryzae bagasse and Paranthaman
35°C 60.5 U/g/min
MTCC rice straw et al., (2008)
Incubation time -
(mixed)
72 hrs
Temperature –
Paranthaman
Redgram 35°C
A.niger 43 U/g/min et al., (2009
husk Incubation time -
b)
96 hrs
pH - 5.5
Sugarcane Temperature -
beggasse 30°C Paranthaman
A.oryzae
and Incubation time - 121 U/g/min et al.,
MTCC 1122
ricestraw 72 hrs (2009 c)
(mixed) Tannic acid conc-
0.06%
pH – 5.5 Manjit
Temperature - et al.,
P.atramen
Amla 28°C 170.75 U/gds (2011)
tosum KM
Incubation time -
96 hrs
pH - 6
Temperature -
Tamarind
30°C
A.niger ATCC seed powder Sabu et al.,
Incubation time - 13.03 IU/g
16620 & Palm (2005)
96 hrs
kernel cake
Tannic acid
conc.-5%
pH-5.5
Sugarcane
Temperature -
beggasse
A.oryzae 30°C Paranthaman
and rice 7.8 U/g/min
MTCC 634 Incubation time- et al., (2010)
straw
72 hrs
(mixed)
pH -5
Amla Ber Temperature -
Jamun 25°C Manjit
A. fumigates 174.32 U/g
Jamoa Incubation time et al., (2008)
Keekar 96hrs
28
Process Enzyme
Strain Substrate Reference
Conditions Activity
pH -5.5
Temperature -
Lokeswari
A. niger Tannic acid 35°C 120U/ml
et al., (2007)
Incubation time -
36hrs
pH -5.5
A. flavus Temperature - 16 U/g/min
Paranthaman
P.chrysogenum Paddy straw 30°C 17 U/g/min
et al., (2010)
T.viride Incubation time - 19 U/g/min
96hrs
pH -6 Anwar
Temperature - et al., (2009)
A.niger Wheat flour 10.8U/ml
50°C
Paddy husk pH -5.5

4.14 U/g/min
Rice bran Temperature -
A.oryzae 14.4 U/g/min Paranthaman
Millet husk 35°C
MTCC 7.41 U/g/min et al., (2009)
Groundnut Incubation time -
11.4 U/g/min
shell 96hrs
Tannic acid conc -
Wheat bran 0.6%
Palm kernel Temperature - 0.35U/gds
Lactobacillus cake 35°C 0.51 U/gds Sabu et al.,
species Tamarind Moisture content - 0.05 U/gds (2006)
seed 44ml 0.21 U/gds
Coffee husk Incubation time -
60 hrs.
Wheat bran pH -5.5
Saw dust Temperature - 3.95 U/g
Rice bran 30°C 1.87 U/g
A.aculeatus Banerjee
Sugarcane Incubation time - 2.93 U/g
DBF9 et al., (2007)
pith 72hrs 1.32 U/g
Rice straw Tannic acid conc. 1.39 U/g
dust -5%
pH -5.5
Temperature -
Cashew 30°C
0.146 Rodrigues
A.oryzae apple Incubation time -
U/gds/hr et al., (2008)
baggasse 48 hrs
Tannic acid conc.
-2.5%
Initial water
Cashew
content -60ml Rodrigues
A.oryzae apple 0.56U/gds
Tannic acid conc. et al., (2007)
baggasse
-2.5%
29
2.6 METABOLIC REGULATION OF TANNASE PRODUCTION

Depending on the strain and fermentation conditions, the tannase can be
produced either constitutively or by substrate induction. Knudson (1913) reported
that tannase production only occurs in the presence of tannic acid, resulting in the
formation of gallic acid and glucose as final products. Seiji et al., (1973) observed
tannase production when the microorganism was grown on glucose as sole carbon
source. Bradoo et al., (1997) showed that Aspergillus japonicus produced tannase
constitutively when grown in a simple culture medium with simple or complex
sugars, but the production of the enzyme was doubled when grew up with tannic
acid as sole carbon source.
Bajpai and Patil., (1997) studied the effect of different substrates such as
gallic acid, pentagalloyl glucose, methyl gallate, and pyrogallol as inducers of the
tannase activity in four species of filamentous fungi, viz., Aspergillus niger, A.
fischerii, Fusarium solani, and Trichoderma viride. They reported that only the
species that were able to grow in the presence of pyrogallol produced the enzyme.
They further observed that each species responded differently to each substrate. A.
fischerii was induced mostly by gallic acid, F. solani by gallotannin, and T. viride
by methyl gallate.
The regulation mechanism of TAH is still unclear, and there exist some
controversies about the specific role of some compounds in the induction and
repression of its expression. It is generally accepted that tannic acid cannot act
directly as an inducer; the molecule is very large and reactive to penetrate the cell
membrane of microorganisms. Gallic acid, which has been used as inducer for the
production of tannase (Bajpai and Patil., 1997), has also been linked to its
regulation (Bradoo et al., 1997). This suggests that the production of TAH is
induced by intermediate compounds produced during the hydrolysis of tannins due
to the action of constitutively produced tannase.
The regulation mechanisms of tannase are very different in SSF and SmF.
Aguilar et al., (2001a,b) studied the induction and repression patterns of production
of tannase in both systems. They found that the addition of tannic acid at
concentrations higher than 25 g/L strongly inhibited the production of tannase in
30
SmF, whereas in SSF, the enzyme was produced in tannic acid concentrations up
to 200 g/L. Furthermore, the addition of small amounts of glucose (12.5 g/L)
increased the activity titles of SSF systems, but there was very less effect in SmF.
Higher concentrations of glucose resulted in a strong catabolite repression in SmF
but had a less effect on SSF. They further reported that the tannase production was
lower than baseline levels of activity with gallic acid as sole carbon source.
2.7 TANNASE RECOVERY

Recovery of the enzyme depends on the production system used and the
time of extraction. In SSF systems, the recovery of tannase is easier due to the fact
that it is extracellular. The addition of two or three volumes of extracting agent
(distilled water or buffer) to mix and compress facilitates the extraction of the
enzyme. However, in the case of the SmF, the localization of enzyme depends on
the time of cultivation. In general, at the beginning of fermentation, the enzyme
was found to be intracellular and this implies that the recovery involves cell
disruption. The cell disruption can be achieved by enzymatic treatment or
mechanical breakdown with mortar or a homogenizer. The recovery is simple;
after cell disruption, it can be achieved by removing cell debris by filtration or
centrifugation. However, at the moment of maximal production, over 80% of the
enzyme remains bound on the mycelium, hindering its extraction ( Barthomeuf et
al., 1994; Bhardwaj et al., 2003).
2.8 TANNASE PURIFICATION

Enzyme purification is performed to increase their catalytic power, improve
stability, or prevent unwanted reactions. There are numerous studies on total and
partial tannase purification. Common protocols of two or three steps include
fractional precipitation (with salts, organic solvents, tannic acid, or pH), ion
exchange or gel permeation chromatography. Other less common steps are affinity
chromatography ( Beverini and Metche., 1990) and preparative scale isoelectric
focusing ( Ramírez-Coronel et al., 2003). The purification process is affected not
only by the type of method used but also by the order in which they are combined.
31
For example, Bhardwaj et al., (2003) increased the yield from 2.7% to 51% on the
purification protocol for the tannase from A.niger van Tieghem by simply inverting
the order of the steps previously used by Sharma et al., (1999).
Enzymes products are available as crude, dried preparations, dilute or
concentrated liquids, or purified solids. Fig.2.8.1 provides a general process
recovery scheme for enzyme derived from animal, plant, surface, or submerged
fermentations. The former source requires immediate pretreatment to release
enzyme into an extracting buffer, followed by the appropriate solids removal steps
when liquid or purified products are required. A more detailed process recovery of
a plant enzyme requires the necessity of good mixing at a low temperature to
maximize initial extraction. The two serial centrifuges perform a solids
fractionation, removing large particles first by scroll centrifugation so that the
more expensive, higher rpm bowl centrifuge is not clogged with large particles.
Subsequent acidification shift pH sufficiently to precipitate much originally soluble
protein, provided a sufficient residence time is allowed in the cooled holding coil
to form a centrifugal precipitate. A disk centrifuge removes this protein precipitate;
a second acidification and holding coil precipitate the desired protein, recovered as
wet solid from the second disc centrifuge. Thus recovery of a plant enzyme
contains two instances where similar or identical processes are placed serially to
carry out a fractionation, first of solids by centrifugation and second by
acidification/ precipitation.
As subsequent smaller scale operations occur in protein purification,
recovery steps may logically shift from continuous to batch as shown in Fig.2.8.2
for enzyme production. Note additional steps to enhance enzyme yield:
(1) repeated washing of biomass (2) two stage ultra filtration to carry out
sequential 5 fold volume reductions, (3) a switch from continuous to batch ultra
filtration to effect a further 40-fold volume reduction.
32
Fig.2.8.1 Preparation of Commercial Enzymes
33
Fig.2.8.2 Extracellular Enzyme Recovery
34
2.9 TANNASE MOLECULAR EXPRESSION

Molecular biology techniques are being employed for better understanding
of tannase and improvement of the yield production. Hatamoto et al., (1996)
cloned and sequenced the gene from tannase of A.oryzae and reported the absence
of introns in its structure. They found that the gene encodes for a sequence of 588
amino acids with a signal sequence of 18 amino acids and a molecular weight of 64
kDa, approximately. This research group hypothesized that the tannase consists of
two subunits 30 kDa and 33 kDa linked by a disulfide bond. The gene of tannase
was transcribed as a single polypeptide chain, after which the signal of 18 amino
acids was cut, and the polypeptide chain was divided in two subunits. It was
reported that the single polypeptide chain was cut by KEX-II like protease in two
amino acid chains. They concluded that native tannase consisted on four pairs of
two subunits, forming a heterooctamer with molecular weight around 300 kDa.
Cerda-Gomez et al., (2006) used conserved sequences of tannase gene from
different species of the genus Aspergillus to design a set of primers (Tan 1 and Tan
2) and used it to amplify a DNA fragment of 435 bp by PCR from four different
species of Aspergillus sp. The amino acid sequence of the tannase gene from
A. niger showed 71% of identity and 10.19% of similarity with A. oryzae. Zhong et
al., (2004) cloned and expressed the tannase gen from A.oryzae in the
methylotrophic yeast Pichia pastoris and obtained large quantities of extracellular
tannase (7,000 U/L) in a short period of incubation (96 hr) in a SmF fed-batch
system. The tannase was purified to homogeneity by a two-step protocol to achieve
a purification factor close to 3.5 with a yield of 51%. Recombinant tannase has
31% more sugar molecules than the native enzyme (22.7%). Although the native
and recombinant enzymes have different degrees of glycosylation, both presented
similar specific activity.
On the other hand, Curiel et al., (2009) reported the production and
purification of a recombinant Lactobacillus plantarum expressed in E. coli. The
tannase gene was inserted with an aminoterminal His-tag that allowed the
convenient purification of the native protein directly from the crude cell extracts.
35
In fact, using this methodology, they were able to obtain large amounts of pure
tannase (17 mg/L) by a one-step affinity procedure.
2.10 MECHANISM OF ACTION

Although the tannins are known as protein precipitants, the tannase acts
over these compounds hydrolyzing the ester bonds formed between galloyl groups
and a polyhydroxy alcohol or the depside link between two galloyl groups (Aguilar
and Gutierrez-Sánchez., 2001; Kasieczka-Burnecka et al., 2007). It has been
proposed that the tannase has a depsidase and esterase activity, and this specificity
depends on the culture conditions (Haslam and Stangroom 1966; Beverini and
Metche., 1990; Farías et al., 1994).
TAH catalyzes the complete hydrolysis of tannic acid to gallic acid and
glucose. The intermediates in the reaction are 1,2,3,4,6-pentagalloylglucose,
2,3,4,6-tetragalloylglucose, and two types of mono-galloyl glucose (Iibuchi et al.,
1972; Lekha and Lonsane., 1997). Fig. 2.10.1 depicts the hydrolysis of 1,2,3,4,6-
pentagalloylglucose catalyzed by tannase. When the substrate is formed by methyl
ester of gallic acid, the TAH produces gallic acid and methanol (Aguilar and
Gutiérrez-Sánchez., 2001).
Tannase activity is inhibited competitively by gallic acid, pyrogallol,
hydroxybenzoic acid, and di-hydroxybenzoic (Iibuchi et al., 1972). The tannase
inhibition by compounds such as diisopropyl fluorophosphate (Adachi et al., 1971;
Barthomeuf et al., 1994) and phenylmethylsulphonyl fluoride (Sharma et al., 2008)
indicates the presence of a serine residue in the active site. Studies with radioactive
isotopes suggested that the sequence of amino acids in the active site can be
threonine–serine–methionine (Adachi et al., 1971). Depending on their origin,
tannase can be inhibited or stimulated in different magnitude by metal ions. The
addition of Mg+2 increased the tannase activity, but it is still unclear whether if it
acts as a cofactor or if it has a nonspecific effect (Kar et al., 2003; Mukherjee and
Banerjee., 2006; Naidu et al., 2008; Hamdy 2008)
36
Fig.2.10.1 Hydrolysis of 1,2,3,4,6- pentagalloyl glucose catalyzed by tannase

(Aguilar and Gutierrez-Sanchez., 2001)
Fig.2.10.2 Esterase and Depsidase activities of Tannase

Toth and Barsony (1943) reported that gallotannin-decomposing tannase
contains two separate enzymes-an esterase and a depsidase with specificities for
ester linkage and m-digallic acid ester linkages, respectively shown in Fig.2.10.2
37
2.11 APPLICATIONS OF MICROBIAL TANNASES

Over the years, tannase enzyme has been used in several conventional
industrial processes, such as Pharmaceutical, Beer and wine production, cold tea
production, treatment of industrial wastewater, containing tannin material, etc
2.11.1 Pharmaceutical industry
Van de Lagemaat and Pyle., (2005) studied major applications of tannase
enzyme are in the production of gallic acid (3, 4, 5-trihydroxy benzoic acid), which
is synthesized chemically in pharmaceutical industry for production of anti
bacterial drug trimethoprim. Gallic acid is a substrate for the chemical and
enzymatic synthesis of propyl gallate, used as an anti oxidant in fats and oils,
foods, cosmetics, hair products, adhesives and lubricant industries.
2.11.2 Beer and wine production
Rout and Banerjee., (2006) reported the application of tannase enzyme in
fruit industries. In fruit industries tannase is also used for clarification and removal
of unwanted bitterness from the traditional fruit juices such as cashew,
pomegranate, cranberry, raspberry, etc. The presence of high tannin content in
these fruits is responsible for haze and sediment formation, which results from
protein–polyphenol interaction. Tannase applied to remove haze, improves color,
bitterness and astringency of the juice upon storage.
Giovanelli., (1989) showed that tannase from a certain strain of A. flavus
has been shown to dramatically reduce the haze formation in beer after storage.
This implicates tannase in the hydrolysis of wort phenolics which complex with
the other chemicals in the beer mixture and results in the haze formation. Upon
treatment of the stored beer with tannase the potential of haze formation is
dramatically reduced.
Canterelli et al., (1989) reported fifty percent of the color of the wines is
due to the presence of the tannins; however, if these compounds are oxidized to
quinines by contact with the air it could form an undesirable turbidity, presenting
severe quality problems. When the proteins of the beer are in considerably high
quantities an undesirable turbidity is presented by accomplishing with these
38
tannins. The use of tannase can be a solution to these problems. In the manufacture
of beer, the tannase could be used since the tannins are present in low quantities.
2.11.3 Cold tea products
Aguilar et al., (2001) studied the commercial applications of tannase is in
the manufacturing of instant tea. Here it is used to eliminate water – insoluble
precipitates (called “tea cream”). These precipitates are generated in a natural way
when the beverage is cooled at temperatures lower than 4°C and if these are
removed chemically (employing sulfites and molecular oxygen with an alkali), a
great amount of aromatic compounds can be eliminated. Such precipitates are
formed by polymerization of esterified poly phenols and by accomplishment of
caffeine, giving a cold water soluble tea, characterized by a high tannin content of
aromatic compounds and appropriate color.
2.11.4 Animal feed
Aguilar et al., (2001) reported the applications of tannase for animal feed
compliment. However, some cultivators of sorghum present a high content of
tannins. If this cereal is first treated with tannase, tannin content could be
decreased and this type of sorghum could then be used as compliment in animal
diet.
2.11.5 Cell wall digestion
Garcia-Conesa et al., (2001) found that tannase may contribute to plant cell
wall degradation by cleaving some of the cross-links existing between cell wall
polymers. Due to the shortage and high cost of the enzyme, the use of tannase in
large-scale applications is limited at present. It is hoped therefore that the
economic benefits of tannase production can help improve the overall viability of
the process.
2.11.6 Effluent treatment
Van de Lagemaat and Pyle., (2001) studied the applications of tannase in
effluent treatment. Tannery effluents contain high amounts of tannins, mainly
polyphenols, which are dangerous pollutants and cause serious environmental
problems. Tannase can be potentially used for the degradation of tannins present in
39
the effluents of tanneries offering a cheap treatment and removal of these

compounds.
2.12 TANNASE IMMOBILIZATION
As mentioned above, TAH has several interesting applications, but its
industrial use is limited mainly due to economical reasons. In this regard, tannase
immobilization offers several advantages over the utilization of free enzyme, such
as improvement of enzyme stability, reutilization of biocatalyst, ease of product
recovery, and continuous operation in packed bed bioreactors (Schons et al., 2011).
Several authors have reported the immobilization of tannase by different
techniques. Abdel-Naby et al., (1999) compared the immobilization of tannase on
several carriers and different methods such as physical adsorption on ASalumina
and colloidal chitin, ionic binding onto Dowex 50W and DEAE-Sephadex A-25,
covalent binding on chitosan and chitin, and entrapment on polyacrylamide and
Ca-alginate beads. They found that covalent binding to chitosan led to the highest
immobilized activity and immobilization yield. Characterization of biocatalyst
demonstrated that immobilization significantly improved the stability at low pH, as
well as thermal stability of tannase.
Mahendran et al., (2005) reported the immobilization of a Paecilomyces
variotii tannase into Ca-alginate beads and utilized them for tannic acid hydrolysis
in a batch reactor. Immobilized biocatalyst completely hydrolyzed a 1% tannic
acid solution within 6 h and retained 85% of its initial activity after eight cycles.
Hota et al., (2007) immobilized a R.oryzae by the same methodology. They
utilized the immobilized tannase for gallic acid production from several tannin rich
agro-residues (sal seed, fruit of myrobalan, and tea leaf). The biocatalyst reached a
high bio-conversion yield (about 90%) and was stable for seven cycles, since the
immobilized tannase showed an appreciable substrate bioconversion of 60–80% up
to seven cycles.
Curiel and co-workers described the immobilization of recombinant L.
plantarum tannase. The enzyme was immobilized and stabilized by one point and
multipoint covalent immobilization on highly activated glyoxyl agarose.
Biocatalyst obtained by multipoint immobilization were 500- fold and 1,000-fold
40
more stable to thermal and cosolvent inactivation than both the soluble enzyme and
the one point immobilized enzyme, respectively. In addition, the immobilized
biocatalyst preserved more than 95% of its initial activity after 1 month of
incubation under the optimal reaction conditions (Curiel et al., 2010).
Immobilized tannase has been employed for treatment of pomegranate and
myrobalan juice (Srivastava and Kar., 2009, 2010) and tea beverage (Su et al.,
2009), as well as for removal of tannin and associated color from tannery effluent
(Murugan and Al-Sohaibani., 2010). Immobilized tannase has also been utilized
for production of gallic acid esters in non-aqueous media, either by esterification of
gallic acid with the appropriate alcohol (Sharma and Gupta., 2003; Yu et al., 2004)
or by direct trans-esterification of tannic acid in presence of alcohol (Fernandez-
Lorente et al., 2011).
Other interesting approach is the utilization of self immobilized tannase.
Under certain production conditions, tannase remains strongly bound to cells; then,
whole cells or cell debris could be used as natural immobilized biocatalysts. For
example, mycelium-bound tannase from A.niger has been employed to catalyze the
synthesis of propyl gallate in organic solvents (Yu and Li., 2005). Belur et al.,
(2010 b) reported the hydrolysis of tannic acid by whole cells of Serratia ficaria
producing a cell associated tannase. This biocatalyst showed a high tolerance to a
wide range of temperature and pH. The use of naturally self-immobilized tannase
offer several technical and economical advantages for industrial applications such
as the avoidance of costly and time-consuming purification and immobilization
processes.
2.13 OPTIMIZATION OF PROCESS PARAMETERS
The production of tannase was carried out so far only in laboratory scale.
The yield of tannase was found to be less and not economical. Hence several
attempts have been made to improve the yield of tannase enzyme by using cheaper
substrates, higher yielding organisms, mixed cultures and by using mutant cultures
with higher rate of fermentation and with shorter fermentation period.
Hence optimization of process parameters for maximum production of
tannase is essential. The classical method and methods like Plackett–Burman
41
Design could not give accurate results for economical and efficient production of
tannase. Statistical optimization techniques like Central Composite Design (CCD)
through Response Surface Methodology (RSM) are widely used to optimize the
media components and process conditions due to its capacity to interpret the
variables affecting the production accurately.
2.13.1 Classical Method
The conventional method that has been used for optimization is the
“change- one-factor-at-a-time” method in which a single factor or one independent
variable is varied while fixing all others at a specific level may. This may lead to
unreliable results and less accurate conclusions.
2.13.2 Plackett–Burman Design
This study was carried out to screen the medium components with respect
to their main effects and not their interaction effects on maximum enzyme
production. Plackett–Burman, (Plackett and Burman., 1946) a two factorial design
identifies critical chemical and physical parameters required for maximum enzyme
production by screening N variables using N + 1 experiments. Minitab-16 is used
to generate experimental design with similar variables. Two values of each
variable {maximum (+) and minimum (-)} are chosen such that the difference
between the two values (+ and -) is large enough to ensure that the peak area for
the maximum enzyme production is included. The variables are analysed. The
effect of variables on enzyme production is calculated by using the following
equation:
E(xi) = 2 ( ∑M+ - ∑M- ) / N …………(2.1)
where E(xi) is the concentration effect of tested variable, M+ and M- are the tannase
production from the trials where the variable (xi) measured was present at high and
low concentrations, respectively and N is the number of experiments carried out.
Experimental error is estimated by calculating the variance among the dummy
variables as follows
Veff = ∑ (Ed)2 / n …………..(2.2)
Where Veff is the variance of the concentration effect, Ed is the
concentration effect for the dummy variable and n is the number of dummy
42
variables in Eqn (2.2). The Standard Error (SE) of the concentration effect was the
square root of the variance of an effect and the significance level (p-value ) of each
concentration effect was determined by using student’s t-test.
t (xi) = E(xi) / SE …………..(2.3)
Where E(xi) is the concentration effect of tested variable and (SE) is the
Standard Error
2.13.3 Response Surface Methodology
Response surface methodology (RSM) is an effective statistical tool and
widely used in process optimization, which includes experimental design, model
fitting, validation and optimization. An effective statistical design is the basis for
response surface optimization and the reported designs include Central Composite
design.
Central Composite Design
Central Composite Design was used to obtain a quadratic model, consisting
of factorial trails and star points to estimate quadratic effects and central points to
estimate the process variability with enzyme production.
Each factor in this design was studied at five different levels -2, -1, 0, +1,
+2 and a set of 31 experiments were carried out. All the variables were taken at a
central coded value considered as zero. The minimum and maximum ranges of
variables were used. All the experiments were carried out in triplicates and the
average value was taken as the response. The CCD experiment was designed using
the Design Expert Software package (Version 8.0.7.1). The following equation was
used for coding the actual experimental values of the factors in the range of ( −2
to +2):
Where xi is the coded value of the ith independent variable, Xi the natural value of
the ith independent variable, X0 the natural value of the ith independent variable at
the center point, and Xi is the value of step change.
Analysis of the data and generation of three dimensional surface graphs
were done using Design Expert Software package (Version 8.0.7.1). After
43
conducting the experiments and measuring the tannase activity levels, a second
order polynomial equation including interactions was fitted to the response data as
given by Eqn (2.4),
Where Y is the measured response, β0 is the intercept term, βi are linear

coefficients, βii are quadratic coefficient, βij are interaction coefficient and Xi and
Xj are coded independent variables.
The significant terms in the model were found by analysis of variance
(ANOVA) for each response. The goodness of fit of the regression model obtained
was given by the coefficient of determination R2. The statistical significance of the
model was determined by F-test. Since coding of the variables enables direct
comparison of the partial regression coefficients, their significance was determined
by student ‘t’ test and associated probabilities. The interaction effects of variables
on enzyme production were studied by plotting three dimensional surface graphs
against any two independent variables while the third and fourth variables are fixed
at its central level.
The second degree polynomial equation was maximized by a constraint
search procedure using the Response optimizer of the Minitab software to obtain
the optimum levels of the independent variables and the predicted maximum
enzyme activity. The predicted enzyme activity was compared with the
experimental values. Validation of the experimental model was tested by carrying
out the batch experiments under optimal operating conditions. All the experiments
were repeated thrice, and the results were compared.
2.14 ARTIFICIAL NEURAL NETWORKS
Artificial Neural Networks (ANN) are a well-known mathematical tool
widely used and tested lately for the problems in industrial enzyme production. Its
advantages are in the ability to handle with nonlinear data, highly correlated
variables and the potential for identification of problems (Mandal et al., 2009).The
fundamental processing element of ANN is an artificial neuron. A biological
neuron receives inputs from other sources, combines them, performs generally a
44
nonlinear operation on the result, and then outputs the final result (Bas et al.,
2007). The basic advantage of ANN is that it does not need any mathematical
model since an ANN learns from examples and recognizes patterns in a series of
input and output data without any prior assumptions about their nature and
interrelations (Mandal et al., 2009).
2.14.1 Structure of ANN
An ANN consists of nodes (neurons) with weighted connections between
them. There are some nodes that receive input, some nodes that give output, and
hidden nodes in between. Each node processes all its input, for example by
summing it up and running the sum through a function, and propagates its result to
its connected nodes until an output is given at some output node(s). Fig.2.14.1
represents the topology of a simple example ANN.
Input nodes receive input data set or information and pass it along to the
hidden nodes through weighted connections. The received signal is processed in
the hidden nodes and sent along weighted connections to output node(s) which
further process the signal and produce the final output ( Jreou et al., 2012)
Fig.2.14.1 Structure of ANN
2.14.2 Properties of ANN

The main property of an ANN is its capability to learn. Learning or training
is a process by means of which a neural network adapts itself to a stimulus by
making proper parameter adjustments, resulting in the production of desired
response. Broadly, there are two kinds of learning in ANNs:
1. Parameter learning: It updates the connecting weights in a neural net.
2. Structure learning: It focuses on the change in network structure (which includes
the number of processing elements as well as their connection types).
45
The above two types of learning can be performed simultaneously or separately.

Apart from these two categories of learning, the learning in an ANN can be
generally classified into three categories as:
· Supervised Learning
· Unsupervised Learning
· Reinforcement Learning
Supervised Learning
In supervised learning, each input vector requires a corresponding target vector,
which represents the desired output. The input vector along with the target vector
is called training pair. The network here is informed precisely about what should
be emitted as output. It is assumed that the correct “target” output values are
known for each input pattern.
Unsupervised Learning
In unsupervised learning, the input vectors of similar type are grouped without the
use of training data to specify how a member of each group looks or to which
group a number belongs.
Reinforcement Learning
In this learning process, only critical information is available, not the exact
information. The learning based on this critic information is called reinforcement
learning and the feedback sent is called reinforcement signal. (Pratap et al., 2013)
2.14.3 Types of ANN
Different types of ANN are known, Kohonen, counter-propagation (CP), back-
propagation ANN, Like in the biological neural network, the artificial ANN has an
interconnection of neurons with three vital components: i) node character which
controls signals i.e. the number of inputs and outputs, the weights and activation
function associated with the node, ii) network topology defining how nodes are
organized and connected and iii) learning rules for the initialization and adjustment
of weights. There are two groups of ANN, supervised and unsupervised, which
differ in the strategy of learning. In unsupervised learning, the input data is
organised and processed without reference to the target, whereas in supervised
learning, both the input and target (output) are used.
46
· Kohonen ANN is an example of unsupervised learning, where no

referential (output) data are used in training of the network, and the
algorithms used are excellent for establishing the relationship among
complex sets of data.
· Counter-propagation ANN represents an up-grade of Kohonen ANN and is
based on two-step learning procedure, unsupervised in the first step, and
supervised in the second. CP-ANN is the most suitable method for
classification of data, but can be used also as a method for developing
predictive models for new objects of unknown properties.
· Back-propagation ANN is another example of supervised learning, where
one or more target values are predicted from input data, meaning that both
inputs and outputs should be known for the training dataset. A special type
of ANN is radial basis function network which ordinarily does not involve
the training of network, but is determined using a certain transformed
function. However, the majority of algorithms work according to an
iterative principle, which is similar to training of the network (Maja
Prevolnik et al., 2012).
47

Chapter-2 Literature Review: 2.0 Sources of Tannase Microbial Source

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Chapter-2 Literature Review: 2.0 Sources of Tannase Microbial Source

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Optimization of Tannase Production from Agro industrial wastes-Kinetics and Modeling

A great variety of bacteria, Klebisella pneumonia (Descamps et al., 1983),

2.1 TANNASE SUBSTRATES

Tannins are naturally occurring water-soluble polyphenolic compounds

mollisima) tannin and quebracho (Schinopsis lorentzii) tannin (Lewis and

Fig.2.1.1 Classification of Tannins ( Khanbabaee and Van Ree., 2001)

Table 2.1 Sources of Tannin

2.2 BIODEGRADATION OF TANNINS

Fig.2.2.1 Gallotannin enzymatic biodegradation pathways (Mingshu et al., 2006)

2.2.1 Bacterial Degradation

In Aspergillus niger, oxygenase converts gallic acid to unstable

2.3.1 Optimum temperature and temperature stability

The temperature dependence of many enzyme-catalyzed reactions can be

fermentation with wheat flour as a substrate and obtained a maximum tannase

Table 2.3.1 Physicochemical Properties of Tannase

Microorganism Optimum pH, Other properties Reference

Aspergillus pH 5.0, 60°C Stable at pH Both intracellular Banerjee

Penicillium pH 5.0, 50°C Active at pH Mol.wt- 2 kDa Sharma

Pencillium pH 5.0-6.0 Stable at Km = 0.48 x 10-4 M Rajakumar

Bacillus cereus pH 4.5, 40°C Stable at Enzyme is salt Mondal

Lactobacillus pH 5.0, 30°C Stable at Enzyme activity Rodríguez

Lactobacillus pH 8.0, 40°C Active at The enzymatic Curiel et al.,

Paecilomyces pH 6.0, 40°C Stable at Mol.wt- 45 kDa Mahendran

A.oryzae pH 5.5, Stable at Molecular weight- Iibuchi et

2.4. STRUCTURE OF TANNASE

Enzyme name : Tannase

Fig.2.4.1 Structure of Tannase

2.5 PRODUCTION OF TANNASE

concentration, inducer concentration, temperature, pH, dissolved oxygen

The summary of the production of tannase enzyme using various microorganisms

Table.2.5.1 Tannase Production by Submerged Fermentation

2.5.2 Tannase Production in Solid State Fermentation

Aguilar et al., (2001) reported the production of tannase using Aspergillus

Kannan et al., (2012) studied production of tannase using Lactobacillus

The production of tannase using various microorganisms by solid state fermentation

Paddy husk pH -5.5

2.6 METABOLIC REGULATION OF TANNASE PRODUCTION

2.7 TANNASE RECOVERY

2.8 TANNASE PURIFICATION

Fig.2.8.1 Preparation of Commercial Enzymes

Fig.2.8.2 Extracellular Enzyme Recovery

2.9 TANNASE MOLECULAR EXPRESSION

2.10 MECHANISM OF ACTION

Fig.2.10.1 Hydrolysis of 1,2,3,4,6- pentagalloyl glucose catalyzed by tannase

Fig.2.10.2 Esterase and Depsidase activities of Tannase

2.11 APPLICATIONS OF MICROBIAL TANNASES

the effluents of tanneries offering a cheap treatment and removal of these

Where Y is the measured response, β0 is the intercept term, βi are linear

Fig.2.14.1 Structure of ANN

2.14.2 Properties of ANN

The above two types of learning can be performed simultaneously or separately.

· Kohonen ANN is an example of unsupervised learning, where no

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