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).