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TRANSCRIPT
Chapter 1.2
Review of Literature
9
1.2 REVIEW OF LITERATURE
1. 2. 1. Tannins
The name ‘tannin’ is derived from the French word ‘tannin’ (tanning
substance) and is used for a range of natural polyphenols. Tannins have relatively
high molecular weight and have the ability to combine strongly with
carbohydrates and Proteins. They were among the first plant natural products to be
utilized industrially, the process of tanning (water proofing and preserving) animal
hides to make leather. High tannin concentrations in nearly every part of the plant,
in the bark, wood, leaves, fruit, roots, and seed. Frequently an increased tannin
production can be due to some sickness of the plant. Therefore, it is assumed that
the biological role in the plant of many tannins is related to protection against
infection, insects, or animal herbivory. (Khanbabaee and Ree 2001). The tannins
appear like as light yellow or white amorphous powders or shiny, almost
colorless, loose masses, with a characteristic strange smell and astringent taste
(Nair et al. 2015). Tannins are oligomeric compounds having free phenolic groups
and complex with proteins, starch, cellulose and minerals. Tannins are present in
both flowering and non-flowering plants of the plant kingdom. Tannins found in
several plant species like Acacia spp, Sericea spp etc. (Hassanpour et al. 2012)
Tannins are water soluble polyphenolic compounds have the ability to bind
with protein that forms soluble or insoluble tannin-protein complexes. It has high
molecular weight ranging from 0.5kDa to 0.3 kDa (Hassanpour et al. 2012).
Tannins are classified into two main groups based on their chemical structure and
properties. First group of tannin is called hydrolysable tannins and the second
10
group is called condensed tannins. Ferreira et al. (1999) reported only two classes
of tannins, namely: (i) condensed tannins and (ii) Complex tannins.
Tannins are antimicrobial agents. Most of the microorganisms cannot
tolerate its polyphenolic nature. Only a few of them can degrade tannic acid and
utilize it as nutrient (Lekha and Lonsane, 1997).
Fig1.2.1.Classification of Tannins.
According to Aguilar et al. (2007) tannins are divided into four major
groups: gallotannins, ellagitannins, condensed tannins, and complex tannins
(Fig.1.2.1). Gallotannins are all those tannins in which galloyl units or their meta-
depsidic derivatives are bound to diverse polyol-, catechin-, or triterpenoid units.
Gallotannins are characterized by the presence of several molecules of organic
11
acids, such as Gallic, digallic, and chebulic acids, esterified to a molecule of
glucose. In the other hand Ellagitannins are those tannins in which at least two
galloyl units are C–C coupled to each other, and do not contain a glycosidically
linked catechin unit. Have a building block of ellagic acid units linked to
glucosides. To maintain its binding capacity, gallotannins, and ellagitannins must
have more than two acidic unit constituents esterified to the glucose core.
Ellagitannins are more stable than gallotannins. Condensed tannins are all
oligomeric and polymeric proanthocyanidins formed by linkage of C-4 of one
catechin with C-8 or C-6 of the next monomeric catechin (Ramirez-Coronel et al.
2004).Complex tannins are tannins in which a catechin unit is bound
glycosidically to a gallotannin or an ellagitannin unit. It can be generated through
reactions between gallic or ellagic acids with catechins and glucosides.
Hydrolysable tannins are composed of esters of gallic acid (gallotannins)
or ellagic acid (ellagitannins) with a sugar core which is usually glucose (Bhat et
al.1998). They can occur in wood, bark, leaves, fruits and galls. Major commercial
hydrolysable tannin sources are Chinese gall (Rhussemialata), sumac
(Rhuscoriaria), Turkish gall (Quercusinfectoria), tara (Caesalpiniaspinosa),
myrobalan nuts (Terminaliachebula) and chestnut (Castanea sativa) (Bhat et al.,
1998). Hydrolysable tannins are readily hydrolyzed chemically by acidification or
biologically by tannase.
1.2.2 Tannase
Tannase (tannin acyl hydrolase) transforms the gallate esters of tannins
and other phenolic compounds, such as epigallocatechingallate, into Gallic acid
12
(GA). GA can later, decarboxylated by gallate decarboxylase to yield Poly gallate
(PG) as end product of tannin metabolism. (Jiménez et al. 2014) (Fig.1. 2.2).It is
generally understood that tannase catalyzes the hydrolysis of tannic (nonagalloyl
glucose) into gallic acid and the molecule of glucose in the ratio of 9:1. And the
mechanism of intermediary compounds is vague. Tannase hydrolyzes other
substrates such as methyl gallate, propyl gallate, digallic acid, epicatechingallate,
and epigallocatechingallate-and release gallic acid. (Fig.1.2.3) (Curiel et al 2009;
Lu and Chen 2007). Iibuchi et al. (1972) analysed some intermediary compound
formed by the hydrolysis using thin layer chromatography. Tannase fully
hydrolyse tannic acid to form glucose and gallic acid through 2, 3, 4, and 6,-
tetragalloyl glucose and form two kinds of monogalloyl glucose and free gallic
acid. This is evident from the fact is that detected the same products in the
hydrolysate of 1,2,3,4,6,-pentagalloylglucose and that gallic acid of
methyl.m.digallate during the degradation pathway (Fig1.2.4). In short tannase is
a group of esters and depsidase and having more specificity to the substrate. It is
relies on the source and methods used for its production and isolation. Teighem
accidentally discovered this unique enzyme and states the formation of gallic acid
when two fungal species were exposed to an aqueous solution of tannins. The
fungal species were afterwards identified as Penicillium glaucum and Aspergillus
niger (Lekha and Lonsane, 1997).
13
Fig 1.2.2 Hydrolytic pathway of tannic acid by tannase.
Fig 1.2.3 Mechanism of action
14
Fig 1.2.4 Esterase and depsidase activity
The main disadvantage of this production of the enzyme is high production
cost. Only a limited number of companies are produced tannase in India.
1.2.3 Microbial source of Tannase
Microorganism is an essential factor for the production of enzyme
industrially. (Table 1.2.1), Microbial sources are chosen for its industrial
production. Besides for large amount enzyme production, fermentation method is
preferable which can be controlled more easily (Lekha and Lonsane 1997. It is
clearly understood that tannins inhibit the growth of many microorganisms, but
there are species that have developed mechanisms to degrade and use them as sole
carbon source. These mechanisms include the production of tannase and other
related enzymes (Banerjee and Pati 2007). It was earlier stated that only a few
microorganisms are able to produce tannase. However, it has been identified that
more than 70 species produce this enzyme, and the number keeps growing as a
15
result of the continuing search for new sources of this enzyme (Belur and
Mugeraya 2011). The production and applications of tannase have been
extensively studied, and investigations related to strain isolation and
improvement, process development, and application of tannases has resulted in a
great number of scientific publications and patents. According to Yamada et al.
(1968) the enzyme was mainly found intracellularly although the culture broth
also contained the enzyme. It was proved that, the growth studies that tannase
enzyme was an inducible enzyme (Gupta et al. 1997; Mattiason and Kaul1994).
To utilize glucose, the organism may synthesize more tannase by which ester and
depside bonds are hydrolyzed and glucose is available for the organism
(Mahapatra et al. 2009).Table 1.2.2 describes some patents regarding tannase
production and application.
Table 1.2.1 Microbial source of tannase
Bacteria Reference
Streptococcus bovis
Streptococcus gallolyticus
Lactobacillus plantarum
Lactobacillus paraplantarum
Lactobacillus pentosus
Lactobacillus acidophilus
Erwiniacarotovora
Belmares et al (2004)
Sasaki et al (2005)
Ayed and Hamdi (2002);
Kostinek et al (2007)
Nishitani and Osawa (2003);
Nishitani et al (2004).
Nishitani et al (2004);
Kostinek et al (2007)
Nishitani et al (2004);
Sabu et al (2006).
Muslim et al. (2015)
16
Fungi
Aspergillus niger
Aspergillus awamori
Aspergillus fumigates
Aspergillus versicolor
Aspergillus flavus
Aspergillus foetidus
Aspergillus terreus
Aspergillus tubingensis
Penicillium chrysogenum
Penicillium variable
Penicillium glaucum
Penicillium crustosum
Penicillium glabrum
Paecilomyces variotii
Penicillium montanense
Bradoo et al (1996); Rana and
Bhat(2005); Cruz-Hernandez et al
(2006);Trevino- Cueto et al (2007);
Muruganet al (2007); Viswanath et al
2015.
Bradoo et al 1996); Mahapatra et al
(2005)
Beena et al (2010).
Batra and Saxena (2005).
Batra and Saxena (2005).
Yamada et al. (1968).
Banerjee et al. (2005)
Nuero and Reyes (2002)
Malgireddy et al. (2015).
Xiao et al (2015)
Rajkumar and Nande (1983)
Batra and Saxena, (2005)
Lekha and Lonsane (1997)
Batra and Saxena (2005)
Batra and Saxena (2005)
Van de Lagemaat and Pyle(2005)
Mahendran et al (2005); Battestin and
Macedo (2007a). Lima et al. (2014)
Yeast
Rhizopus oryzae Hadi et al (1994); Purohit et al (2006).
17
Table 1.2.2 Selected patents on tannase application.
Year Assignee Title Patent
2001 Quest
International
Nederland
Process for the production of beer
having improved flavour stability.
EP 1122303
2002 Unicafe Inc. Tea extracts stabilized for long-term
preservation and method of
producing
same.
USP 6,365,219
2002 Purdue
Research
Foundation
Pharmanex,
Inc.
Tea catechin formulations and
processes for making same.
USP 6,428,818
2004 University of
South Florida
Vasodilating compound and method
of use.
USP 6,706,756
2004 Purdue
Research
Foundation
Compositions based on vanilloid-
catechin synergies for prevention
and
treatment of cancer.
USP 6,759,064
2004 Lipton,
division of
Conopco,
Inc.
Cold brew tea.
USP 6,780,454
2004 Kyowa Hakko
Kogyo Co.,
Ltd.
Process for purification of
proanthocyanidin oligomer
USP 6,800,433
2004 Lipton,
division of
Conopco,
Inc.
Cold water infusing leaf tea.
USP 6,833,144
2006 Nestec S A Soluble coffee product.
EP 1726213
2006 Eisai Co.Ltd Diagnostic agent and test method
for colon cancer using tannase as
index.
USP
7,090,997
2006 Unilever
Bestfoods
Black tea manufacture.
USP 7,108,877
2007 Eisai RandD
Man Co. Ltd.
Novel tannase gene and protein
thereof.
EP 1837400
18
2008 ProbeltePharma
S A
Process for preparing pomegranate
extracts.
EP 1967079
2008 Novozymes,
Inc.
Methods for degrading
lignocellulosic materials.
USP 7,354,743
2008 The Procter and
Gamble
Company
Foam-generating kit containing a
foam-generating dispenser and a
composition containing a high level
of surfactant
USP 7,402,554
2008 Novozymes,
Inc.
Methods for degrading or
converting plant cell wall
polysaccharides
USP 7,413,882
2009 Kirin Brewery Method of enzymatically treating
green tea leaves.
EP 2036440
2009 Kao Corp. Beverage packed in foam container EP 2036446
2009 Kao Corp. Green tea drink packed in container. EP 2098121
2009 Colgate-
Palmolive Co.
Antiplaque oral composition
containing enzymes and
cyclodextrins.
USP 7,601,338
2009 Novozymes,
Inc.
Methods for enhancing the
degradation or conversion of
cellulosicmaterial
USP 7,608,689
2010 Kao Corp. Process for producing purified tea
extract
EP 2225952
2010 University of
California
Method for lowering blood pressure
in prehypertensive individuals
and/or individuals with metabolic
syndrome.
USP 7,651,707
2010 J.M. Huber
Corporation
High-cleaning silica materials and
dentifrice containing such ones.
USP 7,670,593
2010 Novozymes,
Inc.
Polypeptides having cellulolytic
enhancing activity and nucleic acids
encoding
the same.
USP 7,741,466
2010 Constellation
Brands, Inc.
Grape extract, dietary supplement
thereof, and processes therefore
USP 7,767,235
2011 Taiyo Kagaku
Co., Ltd
Composition for inhibiting
thrombosis
USP 7,914,830
2011 Danisco US
Inc.
Polyol oxidases. USP 7,919,295
19
1.2.4 Tannase production
Industrial production of tannase is carried out by employing Aspergillus sp
via solid-state fermentation, modified solid-state fermentation and submerged
fermentation. Mode of fermentation selected depends on strain and culture
conditions. SSF has received popularity for production of tannase and other
enzymes in recent years due to several advantages such as increased titer,
enhanced stability towards temperature and pH and cost effectiveness. Tannase
synthesis is known to get induced by phenolic compounds such as gallic acid,
pyrogallol, methyl gallate and tannic acid (Bajpai and Patil 1997). The induction
mechanism has not been clearly depicted, however there is some argument related
to the role of hydrolysable tannin constituent as related to the synthesis of tannase
(Deschamps et al. 1983; Aguilar et al. 2001a).
1.2.4.1 Submerged Fermentation
Submerged liquid fermentations techniques are widely used for the
production of enzymes derived from microbes. Submerged fermentation can be
carried out by immersing the microorganisms in a solution containing all the
nutrients required for its growth. Tannic acid is widely used as a substrate for
tannase production. Studies have been conducted to optimize the production of
tannase enzyme by moulds under submerged culture and to evaluate the
regulatory aspects of tanase production (Bradoo et al. 1997; Bajpai and Patil,
1997).
20
Tannases are induced by tannic acid or by some of its derivatives but the
regulatory mechanism of its production remains imprecise. Huang et al (2005)
studied tannase as a model system to view experimentally the differences in
enzyme regulation mechanism in both culture systems. Induction and repression
patterns of tannase production by A. niger Aa-20 in solid-state (SSC) and
submerged culture (SmC) were developed. Tannic acid and glucose were used as
carbon sources. Induction and repression ratios were obtained with different
concentrations of tannic acid and glucose, respectively. Valonia tannin (an
ellagitannin) as the substrate, and the factors influencing the yield of ellagic acid
and biosynthesis of valonia tannin hydrolase by Aspergillus SHL 6 were studied.
The factors were analyzed such as Valonia tannin concentration, pH, and
temperature, carbon, and nitrogen sources during the fermentation.
Tannase Production by A. nigerHA37 on a synthetic culture medium
containing tannic acid at different concentrations has been studied. Maximal
enzyme activity increased according to the initial concentration of tannic acid.
Tannase production by A. niger HA37 on four fold diluted olive mill waste waters
(OMWW) as substrate,(0.37 and 0.65 EU/ ml respectively). Growth of A. niger
HA37 on OMWW was correlated with about 70 % degradation of phenolic
compounds present in the waste. Then the strain is used to degrade complex
wastewaters which cause environmental damage to aquatic streams (Aissam et al.
2005). Maximum tannase production occurred in the culture broth containing
1-2 % (w/v) tannic acid and 0.05–0.1 % (w/v) glucose. The optimal value of pH,
incubation period, temperature and glucose concentration optima of tannase
21
production were found at 5.5, 36 h, 35°C and 0.5 % respectively (Lokeswari and
Jaya raju, 2007).
Tannase production by A. awamori BTMFW032 (Beena et al. 2010) has
been studied by slurry state condition. Garcinia leaf as a substrate supported
maximal tannase production. Optimization strategy employing response surface
methodology led to nearly 3-fold increase in the enzyme production from 26.2
U/mL obtained in un optimized medium to 75.2 Units/mL in Box Behnken design,
within 18 h of fermentation.
Production of tannase from Aspergillus niger Van Tieghem was studied by
using different Submerged fermentation processes (Abou‐Bakr et al. 2013). The
SmF gave the highest tannase activity by intermittent shaking (298.4 U/50 mL)
than continuous shaking (80.2 U/50 mL) followed by LSF technique (48.6 U/50
mL).
The optimization studies done by varying one parameter while keeping the
others at constant level do not reflect the interaction effects among these variable
employed and this kind of optimization studies do not depict the net effect of the
various factors on the enzyme activity. To solve this problem, optimization studies
are done using response surface methodology (RSM), which is a mathematical
and statistical technique widely used to determine the effects of several variables
and to optimize different biotechnological processes (He and Tan, 2006).
22
1.2.4.2 Solid state Fermentation (SSF)
SSF allows the construction of more compact reactors with less energy
requirements and causing less damage to the environment (Lekhaand Lonsane,
1997 and Viniegra-Gonz´alez et al. 2003). Solid-substrate fermentations are
generally characterized by growth of microorganisms on water-insoluble
substrates in the presence of varying amounts of free water (Mitchell and
Lonsane, 1992). This process is also referred as solid state fermentation (SSF).
Solid-state fermentation can be defined as a fermentation process that takes place
on a solid or semisolid substrate or in a nutritionally inert support. The origin of
SSF can be traced back to bread-making in ancient Egypt. Solid state
fermentations include a number of well-known microbial processes such as soil
growth, composting, silage production, wood rotting and mushroom cultivation.
In addition, other most popular western foods, such as mold-ripened cheese, bread
and sausage, and many oriental foods including miso, tempeh and soy sauce, are
produced using SSF.
Sabu et al. (2005a) used Palm kernel cake (PKC), and tamarind seed
powder (TSP) for the production of tannase under solid-state fermentation.
Aspergillus niger was grown on the substrates without any pretreatment. In PKC
medium, a maximum enzyme yield was obtained when SSF was carried out at 30
°C, 53.5% initial substrate moisture, 33 ·109 spores/5 g substrate inoculum size
and 5% tannic acid as additional carbon source after 96 h of fermentation.
Cruz-Hernandez et al. (2006) evaluated the effect of culture system on the
production of tannase by an Aspergillus niger strain. They found that enzyme
23
production was about four times higher in SSF compared with SmF. These results
are closely related by Aguilar et al. (2001b) with another strain of A. niger. They
get an activity and productivity at least 2.5 times higher in SSF and associated the
low productivity of the SmF to a possible degradation of the enzyme that is not
present in SSF.
Sharma et al. (2007) used different fungal strains for tannase production
from agro waste as substrate. Aspergillus ruber gave maximum enzyme yield
under solid state fermentation using different tannin rich substrates like ber leaves
(Zyzyphus Mauritiana), jamun leaves (Syzygium cumini), amla leaves
(Phyllanthus emblica) and jawar leaves (Sorghum vulgarism). Jamun leaves
becomes the best Substrate for enzyme production under solid-state fermentation
(SSF)at 30 °C after 96 h of incubation. Induction and repression patterns of
tannase production by Aspergillus niger Aa-20 in solid-state (SSC) and
submerged culture (SmC) found that in SSC an increase in tannic acid enhances
the expression of tannase activity than that of glucose as carbon source.
Lekha and Lonsane, (1994) studied with the comparison of the production
of tannase in SSF, SmF, and Liquid Surface Fermentation (LSF) by Aspergillus
niger PKL104 and found that the enzyme production in SSF was about 2.5 and 4.8
times higher than that obtained by SmF and LSF respectively. In addition, the
activity peak reached in SSF was obtained in about half the time required by the
other two systems. Results attained by Rana and Bhat (2005) with another A.
niger strain also showed that the SSF system is better for tannase production; in
that case, the maximum yield achieved in SSF was 1.6 times higher than that
24
obtained by SmF, also tannase produced by SSF was more stable at a wide range
of temperatures and pHs.
The most profitable applications of SSF are in the Oriental and African
countries where SSF processes have been perfected over long periods. SSF
processes are recognized by investigators to be suitable for the production of
enzymes by filamentous fungi since they reproduce the natural living conditions
of such fungi (Rodrıguez-Couto and Sanroman, 2005).
The idea of the tannase production process is to use coffee pulp or coffee
pulp juice as a tannin-rich substrate and achieve direct breakdown of the
hydrolysable tannins present. Previous studies suggested that SSF is advantageous
over conventional submerged fermentation for the productive yield of tannase
which is an inducible enzyme using coffee wastes The developed process could
potentially be used with other tannin-rich agricultural residues such as cassava,
carob bean, wine-grape and tea waste (Lekha and Lonsane 1997).
Aguilar et al. (1999) reported production of tannase under SSC and SmC,
respectively, and the maximum tannase activity expressed intracellularly was also
18 times more in SSC than in SmC, while the extracellular activity was 2.5 times
higher in SSC than in SmC.
Extra and intracellular tannase production by A.niger GH1 studied using
submerged (SmF) and solid-state fermentation (SSF) at different temperatures (30,
40 and 50°C). Different parameters such as initial substrate (tannic acid)
concentration, incubation time and temperature on tannase production in SSF have
25
been studied. A. niger GH1 produced the highest tannase level (2291 U/L) in SSF
at 30°C during the first 20 h of culture at tannic acid concentration of 50 g/l, and
under these conditions and become extracellular condition Cruz-Hernandez et al
2005). Tannase production under SSF by A.nigerAa-20 using gobernadora powder
as the sole carbon source and as an inducer of tannin-degrading enzymes was
evaluated. Tannase production reached values of 1040 Ul/1 at 43 h of culture.
During the first 48 h of culture, the concentration of gallic acid accumulation was
0.33 g/l. It was proved that Gobernadorais a potential source of gallic acid and
tannase production by solid state culture (Trevino-Cueto et al 2007).
Tannase production from newly isolated Aspergillus terrus under solid
state fermentation from wheat bran as a substrate was studied. Tannase production
was achieved with 1.5% Sucrose and 1.75% yeast extract whereas Glucose did not
repress enzyme production but inorganic nitrogen sources showed little negative
impact. The main parameters like, pH of the medium (pH 3.5), moisture content
(60%), incubation time (72 h) and inoculum level (3ml) played essential role in
tannase production.
Xiao et al. (2015) studied tannase production by Aspergillus tubingensis in
solid-state fermentation using tea stalks as a solid support. They used Plackett–
Burman design for initial screening and central composite design with response
surface analysis. Seven tested variables were identified as the most significant
factors for tannase yield. The experimental value of 84.24 units per gram of dry
substrate (U/gds) very much matched the predicted value of87.26 U/gds. Materials
used as supports of SSF for tannase production and tannin-rich materials used as
enzyme inducer in SSC and SmF are presented in Table 1.2.3.
26
Table 1.2.3 Resources used as supports of SSF for tannase production and
tannin-rich materials used as enzyme inducer in SSF.
Natural supports Reference
sugarcane bagasse Lekha and Lonsane (1994); Garcia-Pena et al
(1999)
Wheat bran Malgireddy and Nimma2015; Sabu et
al(2005b), Ma et al 2015
tamarind seed powder Sabu et al (2005a)
palm kernel cake Sabu et al (2005b)
cashew apple bagasse
(Anacardium occidentale)
Banerjee et al (2005)
Fruits of Terminaliachebula Banerjee et al (2005)
pod cover of Caesalpiniadigyna Banerjee et al (2005)
ber leaves (Ziziphus mauritiana) Kumar et al (2007)
jamun leaves (Syzygium cumini) Kumar et al (2007)
amla leaves
(Phyllanthus emblica)
Kumar et al (2007)
jawar leaves (Sorghum vulgaris) Kumar et al (2007)
Garcina Leaves Beena et al (2010)
Creosote
bush leaves (Larreatridentata)
Trevino-Cueto et al (2007)
Cashew testa
(Anacardium occidentale)
Viswanath et al (2015)
Polyurethane foam Ramirez-Coronel et al (1999);
Aguilar et al(2001b)
Van de Lagemaat and Pyle (2001, 2005)
sponge as a synthetic solid (AbouBakr et al 2013).
barbados cherry and mangaba
fruit
Lima et al (2014).
27
1.2.5. Purification and characterization of tannase
Several strategies can be used for tannase concentration or purification and
immobilization after extraction from the biomass (solid state fermentation) or
from the culture medium (submerged fermentation).
The purification process is one of the less developed aspects on tannase.
Most of the published purification protocols consist on multistep procedures able
to obtain a highly purified enzyme but with a low-recovery yield. The common
strategy used for the purification of tannase based on protein concentration
followed by ion exchange and/or gel filtration chromatography (Beniwal et al.
2003; Bharadwaj et al. 2003 and Mahendran et al. 2005).
Tannase has been purified from a variety of fungi like A. flavus (Yamada
et al. 1968), A. oryzae (Iibuchi et al. 1968), Candida sp.(Aoki et al. 1976),
Penicillium chrysogenum(Rajkumar and Nandy, 1983) and A. niger (Barthomeuf
et al. 1994;Viswanath et al. 2015). Based on extracellular and intracellular nature
of enzyme production, culture filtrate as such or mycelia extract after sonication
(Yamada et al 1968) were used as the crude enzyme.
Beverini and Metche (1990) reported acetone precipitation as initial step
for the purification of tannase. Mahapatra et al. (2005) later purified the acetone
precipitated fraction by using G-100 sephadex column. Sharma et.al. (2008)
purified the tannase using ultrafilteration using membrane cartridges of different
molecular weight cutoff followed by gel filtration chromatography using G-200
sephadex column. Initial and final purification step obtained 97% and 91% yield
28
and 5 .0 and 135 fold purification respectively. Costa et al. (2012) purified the
extracellular tannase by using two chromatography techniques, filtration
chromatography using G-150 sephadex column followed by ion exchange
chromatography in a DEAE Sephadex column which allowed the separation of
two isoforms of tannase designated as TAH I and TAH II of which TAHI showed
more than 70% of total tannase activity. Aoki et al. (1976) and Lekha and
Lonsane, (1994) reported failure of ammonium sulphate to precipitate tannase
because of the very low yield. Tannase from A. Awamori MTCC 9299 was
purified using ammonium sulfate precipitation followed by ion exchange
chromatography. Chhokar et al. (2009) obtained purification fold of 19.5 with
13.5 % yield respectively. Tannase precipitation using polymers 1-90 % such as
poly vinyl alcohol, polyethylene glycol and dextran have been reported by Naidu
et al. 2008. Ultra filtration membranes were also used in concentrating the enzyme
recently (Sharma et al. 2007; Marco et al. 2009).
The final step of purification was Gel filtration chromatography. Tannase
being a high molecular weight protein sephadex G-200 was used by most of the
workers (Raj kumar and Nandy 1983; Lekha and Lonsane, 1994; Sharma et al.
2007) An extracellular tannase produced by solid-state cultures of A. niger was
purified to homogeneity from the cell-free culture broth by preparative isoelectric
focusing and by fast protein liquid chromatography (FPLC) using anion-exchange
and gel-filtration chromatography (Ramirez-Coronel et al. 2003). Sephadex G-100
or Sephadex G-100 super fine, and G-50 (Ramirez-Coronel et al. 2003, Viswanath
et al. 2015) and sephacryl S-300 gel filtration (Marco et al. 2009) were also used
to separate tannase from A.niger.
29
Internal sequences were obtained from each of the gel-purified and trypsin
digested tannase forms. The peptide sequences obtained from both forms were
identical to sequences within β-glucosidase from A. kawachii. The purified
tannase was tested for β-glucosidase activity and was shown to hydrolyze
cellobiose efficiently. However, no β-glucosidase activity was detected when the
enzyme was assayed in the presence of tannic acid (Ramirez-Coronel et al. 2003).
Zhong and coworkers (2004) reported that the recombinant tannase from
Aspergillus oryzae expressed in Pichia pastoris was easily purified to
homogeneity. Curiel et al. (2003) described a high-yield protocol for the
purification of a recombinant Lactobacillus plantarum tannase expressed in
Escherichia coli. The protein was cloned containing an affinity hexa-His tag, this
allowed to purify the recombinant tannase directly from the crude extract using a
His-Trap-FF chelating column. In this case recombinant tannase is valuable.
The molecular weight of characterized tannases was found to be in the
range of 50–320 kDa depending on the source. Fungal tannases mostly have been
reported to be multimeric proteins formed by 2 to 8 subunits. Ram´ırez-Coronel et
al. (2003) purified and characterized an Aspergillus niger tannase which is active
in monomeric and dimeric isoforms of 90 and 180 kDa, respectively. Boer and
coworkers (2009) found that tannase from the dimorphic yeast Arxula
adeninivoransis composed homo tetramer with subunits of 80 kDa. Beena et al.
(2010) reported a tannase of A. awamori formed by six identical subunits of 37.8
kDa. Hatamoto et al. (1996) reported that native tannase of A. oryzae consists of
four pairs of two types of subunits (30 and 34 kDa, respectively) linked together
by disulfide bonds, forming a hetero octamer of 310 kDa. Furthermore, all
30
bacterial tannases characterized are monomeric with a molecular weight ranging
from 50 to 90 kDa (Sharma and John, 2011, Iwamoto et al. 2008, Skene and
Brooker 1995).
Niehaus et al (1997) reported that the tannase from the Penduculate oak
exhibited two protein bands that contained esterase activity. After denaturation on
SDS-PAGE they observed only one polypeptide band of molecular mass of 75
kDa,
An A. flavus with molecular weight 192 kDa had 25.4 % carbohydrate
content (Yamada et al. 1968; Adachi et al. 1971). A. niger with a molecular
weight of 186 kDa was reported to have 43 % carbohydrate content (Barthomeuf
et al. 1994; Parthasarathy and Bose, 1976). Whereas A. oryzae tannase with 300
kDa molecular weight had 22.7 % carbohydrate content (Hatamoto et al. 1996;
Abdel-Naby et al. 1999), A. awamori formed by six identical subunits of 37.8 kDa
had 8.02% carbohydrate content (Beena et al. 2010).
The optimum pH range for tannase activity of the preferred fungi was
found to be 3.0 to 8.0. Barthomeuf et al. (1994) reported for A. niger pH optimum
of 5.0–6.0, Batra and Saxena(2005) reported that tannase from A. fumigates and A.
flavusis stable at pH 4.0, and doesn’t show tannase activity at alkaline pH of
8.0.However A. versicolor tannase showed a relatively wider range of pH stability
at pH 8.0 and less stability at pH 3.0 with maximum (100%) at pH 6.0. Sharma et
al. (2008) reported the optimum activity of tannase by P.variable at pH 3.0.
Rajakumar and Nandy, (1983) reported that tannase from Penicillium
chrysogenum showed broad pH dependence with optimum enzyme activity at a
31
pH of 5.0 -6.0, with the enzyme apparently stable at 16°C in a pH range of 4.0 to
6.5. Beena et al. (2010) reported that tannase from A. awamori at pH 2.0. So it can
be concluded that the fungal tannase is an acidic protein and needed an acidic
environment to be active (Mahapatra et al. 2005).
The optimum temperature and range for tannase activity of the selected
tannase producers were evaluated by moving out the reaction at different
temperatures ranging from 30 to 80◦C at their respective optimum pH by different
researchers. Batra and Saxena (2005) found that the functional temperature range
of the tannase is 30–70◦C with optima at 60◦C for A. flavus, A. fumigatus,
A.versicolor and P. variable, whereas A. caespitosum, P.charlesii, P. crustosum
and P. restrictum had an optimum activity at 40◦C. These results are also Sharma
et al. (2008) experimentally bring it to notice that the three different strains of
Penicillium variable showed optimum tannase activity at 50°C.Mahapatra et al.
(2005) reported that the optimum temp for tannase activity was 35°C for A. oryzae
and P.chrysogenum. The optimal temperature previously reported for the maximal
production of tannase in SSF was between 25 and 34oC for A. niger, A. acuelatus,
Lactobacillus sp. and Paecilomyces variotii, (Anwar et al. 2007, Banerjee et al.
2007, Battestin and Macedo (2007a) , Mukherjee and Banerjee (2004), Sabu et al.
(2006).
The effects of metal salts and organic solvents on the activity of tannase
were also studied. Metal salts Mg+2, Mn+2, Ca+2, Na+, and K+ stimulated the
tannase activity, while Cu+2, Fe3+, and Co2+ acted as inhibitors of the enzyme. The
addition of organic solvents like acetic acid, isoamylalcohol, chloroform,
32
isopropyl alcohol, and ethanol completely inhibited the enzyme activity. However,
butanol and benzene increased the enzyme activity (Chhokar et al. 2009).
KM value of tannase for different fungi with tannic acid was different. The
values of kinetic constants (KM and Vmax) depend on the particular substrate
used and the enzyme source. A wide range of values (2×10-5-1.03×10-3M) for KM
and Vmax have been reported for tannases from several microorganisms
(Bhardwaj et al. 2003, Rajkumar and Nandy 1983). Tannase from A. niger GH1
recorded KM and Vmax values of 0.41×10-4 M and 11.03 μmol/min, respectively,
with methyl gallate as a substrate (Marco et al. 2009). The kinetic parameters of
tannase self-immobilized on polyurethane particles were calculated to be 5 mM
and 0.41×10−2 mM/min for KM and Vmax. (Mata-Gomez et al. 2015)
Tannase requires the presence of metal ions to express its full catalytic
activity; so it is important to know about the concentrations of ions for attaining
maximal reaction productivity. The effect of metal ions on tannase activity was
studied (Kar et al. 2003). One mM Mg2+ or Hg+ activated tannase activity. Ba2+,
Ca2+, Zn2+, Hg2+, and Ag+ inhibited tannase activity at 1.0 mM concentration, and
Fe3+ and Fe2+ completely inhibited tannase activity. Ag+, Ba2+, and Hg2+
competitively inhibited tannase activity (Mukherjee and Banerjee, 2005). Among
the anions studied 1 mM Br-or (S2O3)-2 enhanced tannase activity. Tween 40 and
Tween 80 enhanced tannase activity whereas Tween 60 inhibited tannase activity.
Palmitic acid and oleic acid enhanced tannase activity, whereas stearic acid
inhibited tannase activity (Kar et al. 2003). Sodium lauryl sulfate and triton X-100
inhibited tannase activity. Urea stimulated tannase activity at a concentration of
33
1.5 M. Between the chelators, 1mM EDTA or 1,10-ophenanthrolein inhibited
tannase activity dimethyl sulphoxide and β-mercaptoethanol inhibited tannase
activity at 1 mM concentration whereas soybean extract inhibited tannase activity
at concentrations varying from 0.05 to 1.0 % (w/v). Among the nitrogen sources
selected ammonium ferrous sulfate, ammonium sulfate, ammonium nitrate and
ammonium chloride enhanced tannase activity at 0.1 % (w/v) concentration (Kar
et al. 2003; Mukherjee and Banerjee, 2005). The tannase from A. niger was
reported to be inactivated by β-mercaptoethanol (Aguilar and Gutierrez-Sanchez,
2001). No inhibition by EDTA was observed in the case of the tannase from A.
flavus (Yamada et al. 1968). Sabu et al. (2005a) found that, the highest enzyme
activity (3.9 U/ml), after 15-20 min of incubation time, with an activity of KM
was found to be 1.03 mM and Vmax 4.25 mol/min respectively. Subsequently the
enzyme is active over a wide range of pH and temperature; it could find potential
use in the food-processing industry. Tannase from A. Awamori nakazawa
exhibited optimum activity at 35°C and at a pH of 5.0. Urea concentrations higher
than 3M were inhibitory. Increasing concentrations (2%) of sodium lauryl sulfate
(SLS) also led to decrease in activity. Increasing concentrations of ethylene
diamine tetra acetic acid (EDTA) had an inhibitory effect on tannase (Mahapatra
et al. 2005).
A kinetic and thermodynamic study was performed on the esterification of
propyl gallate from gallic acid and 1-propanol by mycelium-bound tannase from
A.niger in organic solvent. A kinetic model of esterification by mycelium-bound
tannase was developed based on the Ping–Pong Bi–Bi kinetic mechanism,
considering not only the effect by substrates and products, but also tannase
34
denaturation. A reasonable quality of fit was observed by fitting experimental rate
data to the kinetic mode with an average correlation coefficient of 0.977. Further
when neglected the inactivation of tannase, the kinetic model fitted the
corresponding experimental data poorly (Yu and Li, 2006).
1.2.6 Genetic characterization
Hatamoto et al. first reported complete sequence of tannase gene in 1996.
They found Aspergillus oryzae tannase gene and is coded by 588 amino acids
sequence with a molecular weight of about 64000 kDa. Native protein Analysis
showed that the protein is a single polypeptide and by post translational
modification it is cleaved into two tannase subunits which is linked by a disulfide.
They concluded mature protein forms a hetero-octamer consisting four pairs of the
two subunits, with a molecular weight of 300 000. Then by structural homology
tannase gene of many organisms has been identified but only a few have been
confirmed at protein level. Hatamoto et al. (1996) cloned and expressed a tannase
gene from A.oryzae in Pichia pastoris and the catalytic activity of this
recombinant enzyme was assayed. With the aid of Saccharomyces cerevisiae
factor, a secretary form of enzyme was made, and a simple purification protocol
yielded tannase in pure form. By fed-batch culture the productivity of secreted
tannase was achieved around 7000 IU/l. Recombinant tannase consisted of two
types of subunits linked by disulphide bonds and have a molecular mass of 90 kDa
(Zhong et al. 2004). A comparison of tannase gene cDNA from A. Oryzae with
genomic DNA sequence for tannase from revealed that no introns were present in
the genes and both the genes proved to be very similar. The tannase gene from A.
35
niger was 1740 bp in in contrast to the tannase gene from A. oryzae being 1767 bp
in size. DNA sequence between these two tannase gene sequences revealed on
nucleotide level. Amino acid alignments revealed that the ORF for A. niger
showed 71.5 % identity and a similarity of 10.19 % sequence for tannase from A.
oryzae. Modification of the peptide sequence for tannase from A. oryzae is
cleaved into two subunits by a KEX-II like protease at position 315 and liberating
two peptide subunits of 30 kDa and 33 kDa in size. Tannase gene from A. oryzae
was PCR amplified anpRS426 containing a PCR amplified PDC1glucose induced
promoter. (Hohmann, 1991).
Newly, Leόn-Galv´an and co-workers (2011) cloned and sequenced the
complete cDNA of a tannase gene from Aspergillus niger and found an ORF was
of 1833 bp. The 5' untranslated (1822 bp) and a 3' UTR (1015 bp). Tannase gene
A. awamori was isolated and sequenced in 2009 (Beena et al. 2010) and found an
ORF of 1,122 bp. Homology studies revealed a higher similarity of the awamori
gene with A. niger than with the A. oryzae gene (77%). Boer et al. (2009)
identified the tannase-encoding gene from the dimorphic Arxula adeninivorans
and has an ORF of 1764 bp and encodes a 587-amino acid protein, preceded by an
N-terminal secretion sequence comprising residues. The deduced acid sequence
was similar to those of tannases from A. oryzae (50% identity) and A. niger (48%).
Noguchi et al. (2010) reported a tannase gene from bacteria. They cloned and
sequenced a novel gene (tanA) from Staphylococcus lugdunensis that encodes a
polypeptide of amino tannase activity. Later, Iwamoto and coworkers cloned and
sequenced the tannase gene from Lactobacillus plantarum (tanLpl). The tanLpl
gene was almost identical to a nucleotide sequence of L. plantarum WCFS1
36
designated as lp2956 (99.6% identity), encoding a hypothetical protein but single
base substitution at four positions and was similar (46.7%) to tanA from
S.lugdunensis (Iwamoto et al. 2008). More recently, the characterization of the
tannase gene from Enterobacter sp. Multiple alignment showed that Enterobacter
sp. tannase is not very much similar to tannase of S. lugdunensis and L.
plantarum, since only 10% and 13% amino acid residues of Enterobacter sp.
Tannase are similar to S. Lugdunensis, L.plantarumtannases, respectively.
Additionally, bacterial tannase genes are not closely related to fungal tannases, the
tannase encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA
by PCR, using as primers oligonucleotide sequences derived from peptides obtained after
tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp,
encoding a 587-amino acid protein, preceded an N – terminal secretion sequence
comprising 28 residues. The deduced amino acid sequence is similar to those of tannases
from Aspergillus identity and putative tannases from A. fumigatus (52 %) and
A.nidulans (50%). The motif (-Gly-X-Ser-X-Gly-), forms part of the catalytic
centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon
source. Supplementation with tannic acid or gallic acid leads to induction of
ATAN1 and accumulation of the native tannase enzyme in the medium. The
enzymes recovered from both wild-type and recombinant strains were essentially
indistinguishable. A molecular mass of 320 kDa was determined, indicating that
the native, glycosylated tannase consists of four identical .The enzyme had a
temperature optimum at 35-40°C and a pH optimum at 6.0. The enzyme was able
to remove gallic acid from both condensed and tannins. Under inducing wild type
strain LS3 secreted around 100 U/l tannase, while the transformant strain, which
37
over expressed the ATAN1 gene from the strong, constitutively active A
adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in
glucose medium in shake flasks (Boer et al. 2009).
1.2.7 Applications of tannase
Tannase has numerous interesting applications in food, feed, chemical, and
pharmaceutical industries etc. (Fig 1.2.5). Mainly tannase are used in the
elaboration of instantaneous tea and the production of gallic acid ester by
depolymerization of tannin-rich materials (Lekha and Lonsane, 1997). But, due to
its hydrolytic and synthetic properties, tannase has several other potential
applications.
Fig 1.2.5. Application of Tannase
38
1.2.7.1 Industrial
1.2.7.1.1 Pharmaceutical industry
Gallic acid has been synthesized chemically, but this chemical synthesis
has been known to be very expensive and not considered always. Gallic acid is
one of the by-product liberated upon hydrolysis of tannic acid with tannase
(Iibuchi et al. 1972). It is used as a synthetic intermediate for the production of
pyrogallols and gallic acid esters. Currently gallic acid is mainly used for the
synthesis of trimethoprim, and for the production and synthesis of propyl gallate,
which is used as an antioxidant in fats and oils (Weetal, 1985).
Fig.1.2.6.Transesterification of tannic acid to propyl gallate in presence of n-
propanol
1.2.7.1.2 Beverage clarification
Fruit juices (pomegranate, cranberry, raspberry, etc.) have recently been
acclaimed for their health benefits, in particular, for their antioxidant properties.
On the other hand, the presence of high tannin content in those fruits is
responsible for haze and sediment formation, in addition to for color, bitterness,
39
and astringency of the juice upon storage. Enzymatic treatment with tannase may
be used to improve the quality of these juices (Aguilar et al. 2007).
Rout and Banerjee (2006) reported the use of tannase for pomegranate
juice debittering. Enzymatic treatment resulted in 25% degradation of tannin,
while a combination of tannase and gelatin (1:1) resulted in 49% of tannin
degradation. This treatment has no negative impact on the biochemical and quality
attributes of the fruit juice. Hydrolysis by immobilized tannase removed up to
73.6% of the tannin present in Indian gooseberry (Phyllanthus emblica) juice.
This enzymatic action reduced the tannin content but increased the gallic acid
concentration with a minimum reduction in vitamin C (only 2%) (Srivastava and
Kar 2009, 2010).
Tannase from A. flavus has been shown to reduce the haze formation in
beer after storage. This implicates tannase in the hydrolysis of phenolics which
complex with other chemicals in the beer mixture and results in the haze
formation. Giovanelli, (1989) showed that upon treatment of the stored beer with
tannase the potential of haze formation was dramatically reduced.
Initially wine was treated chemically to remove the unfavoured phenolics.
Now tannase is being employed to hydrolyse chlorogenic acid to caffeic acid and
quinic acid, which influences the taste of the wine favorably (Chae et al.1983).
1.2.7.1.3 Instantaneous tea elaboration
After water, tea is the second most highly consumed beverage worldwide
(Venditti et al. 2010). It is an infusion obtained from leaves of Camellia
40
sinensisand is consumed by two-thirds of the world’s population (Łuczaj and
Skrzydlewska, 2005). Tea drinking is related with the reduction of serum
cholesterol, prevention of low-density lipoprotein oxidation, and decreased risk of
cardiovascular disease and cancer (Karak and Bhagat 2010). During the
production of tea beverages, hot and clear tea infusions tends to form turbid
precipitates after cooling. These precipitates, called tea cream, are formed by a
complex mixture of polyphenols. Tea cream formation is a quality problem and
may have antinutritional effects (Lu et al. 2009). Tannase can hydrolyze the ester
bonds of catechins to release free gallic acid and water-soluble compounds with
lower molecular weight, reducing turbidity and increasing solubility of tea
beverage in cold water. Thus, tannase has been widely used to hydrolyze tea
cream in the processing of tea (Su et al. 2009).
Enzymatic treatment of tea beverage leads to a better color appearance,
less cream formation, better taste, mouth feeling, and overall acceptance (Lu et al.
2009). Moreover, the hydrolysis of tea phenols epigallocatechingallate and
epicatechingallate to epigallocatechin and epicatechin, respectively, increases the
antioxidant activity of tea beverage (Lu and Chen 2008).
1.2.7.1.4 Animal feed
Tannins are ubiquitous in nature and are widely found in feedstuffs,
forages, fodders, and agro industrial wastes, affecting livestock production
(Krueger et al. 2010). Ant nutritional effect of tannin could be reduced by a
treatment with tannase or tannase producing microorganism. For example, there
are some cultivars of sorghum with high content of tannins. Tannin content could
41
be decreased by an enzymatic treatment, and this material could be used as
complement in animal diet (Aguilar et al. 2001).
Nuero and Reyes (2002) reported the production of an enzymatic extract
containing tannase from mycelial wastes of penicillin manufacture. This
preparation was applied to several flours used as animal feed (barley, bran, maize,
oat, rye, soya, and wheat flour). The enzymatic extract from mycelia waste
released similar amounts of reducing sugars from all flours when compared with a
commercial enzymatic additive used in animal feeding. These clarifications
indicated that tannase-containing preparation has a high potential as supplements
for animal feeding.
1.2.7.1.5 Cell wall digestion
Tannase could add to plant cell wall degradation by cleaving some of the
cross-links existing between cell wall polymers (Garcia-Conesa et al, 2001). Due
to lack and high cost of the enzyme, the use of tannase in large-scale applications
is inadequate. So it can help the economic benefits of tannase production.
1.2.7.1.6 Effluent treatment
Tannery effluents contain high amounts of tannins, mainly polyphenols,
which are dangerous pollutants and cause serious environmental problems (Van
de Lagemaat and Pyle, 2001). Tannase can be potentially used for the degradation
of tannins present in the effluents of tanneries offering a cheap treatment and
removal of these compounds.
42
1.2.7.2 Environmental
1.2.7.2.1 Bioremediation of tannin-contaminated waste water
Kachouri and co-workers (2005) studied the biodegradation and
decolourisation of olive-mill wastewater by using Aspergillus flavus. The
microorganism removed 58% of color and 46% of the chemical demand of
oxygen of the wastewater after 6 days of cultivation. Hence it concluded that, this
degradation with deconcomitant production of tannase, since no lignin peroxidase
nor manganese peroxidase were detected, and laccase activity was much lower
than tannase activity.
Tannins are resistant to microbial attack and known to inhibit the growth
of some microorganisms. The antimicrobial effect of tannin slows down the rate
of biodegradation of soil organic matter (Scalbert, 1991). Polyphenolic
compounds on tannin substrate structure form complex with extra cellular and
intracellular enzymes from biodegradative organisms. The complexation leads to
inhibition of biodegradative enzymes which leads to loss in microbial growth and
increase in bioconversion time taken for decomposition of soil organic matter.
Tannase could decrease the bioconversion time for decomposition of soil organic
matter (Albertse, 2002).
Tannase is also used in the manufacture of sensitive analytical probes for
determining the structure of naturally occurring gallic acid esters (Haslam and
Tanner 1970). In addition, it is incorporated into the manufacture of high grade
43
leather (Barthomeuf et al. 1994) and is used to clean up the hard and acidic
industrial effluent containing tannin materials (Banerjee, 2005).
1.2.7.3. Other Applications
There are other potential applications are in tannase like fuel ethanol
production from agro industrial wastes and Leather industries have gained in
current years. When these feed stocks are pre-treated for delignification, simple or
oligomeric phenolics and derivatives are generated from lignin. These compounds
can inhibit the hydrolysis catalyzed by cellulases. Tannase could be utilized for
degradation of these oligomeric phenolics and, by doing so, mitigate the inhibition
on cellulolysis (Tejirianand Xu, 2011).