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Production and Partial Purification of Tannase FromAspergillus ficuum Gim 3.6Wan-liang Maa, Fen-fen Zhaoa, Qin Yea, Zhen-xing Hua, Dong Yana, Jie Houa & Yang Yanga

a College of Life Science and Technology, Guangxi University, Nanning, ChinaAccepted author version posted online: 15 Aug 2014.

To cite this article: Wan-liang Ma, Fen-fen Zhao, Qin Ye, Zhen-xing Hu, Dong Yan, Jie Hou & Yang Yang (2014): Productionand Partial Purification of Tannase From Aspergillus ficuum Gim 3.6, Preparative Biochemistry and Biotechnology, DOI:10.1080/10826068.2014.952384

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Production and partial purification of tannase from Aspergillus ficuum Gim 3.6

Wan-liang Ma1, Fen-fen Zhao

1, Qin Ye

1, Zhen-xing Hu

1, Dong Yan

1, Jie Hou

1, Yang

Yang1

1College of Life Science and Technology, Guangxi University, Nanning, China

Corresponding author: E-mail: yay1008@163.com

Abstract

A novel fungal strain, Aspergillusficuum Gim 3.6, was evaluated for its

tannase-producing capability in the wheat bran-based solid state fermentation. Thin-layer

chromatography (TLC) analysis revealed that the strain was able to degrade tannic acid to

gallic acid and pyrogallol during the fermentation process. Quantitation of enzyme

activity demonstrated that this strain was capable of producing a relatively high yield of

extracellular tannase. Single-factor optimization of process parameter resulted in high

yield of tannase after 60 h of incubation at a pH of 5.0 at 30oC, 1 ml of inoculum sizeand

1:1 of solid-liquid ratio in the presence of 2.0% (w/v) tannic acid as inducer. The

potential of aqueous two-phase extraction (ATPE) for the purification of tannase was

investigated. Influence of various parameters such as phase-forming salt, molecular

weight of polyethylene glycol (PEG), pH, and stability ratio on tannase partition and

purification was studied. In all the systems, the target enzyme was observed to

preferentially partition to the PEG-rich top phase, and the best result of purification

(2.74-fold) with an enzyme activity recovery of 77.17% was obtained in the system

containing 17% (w/w) sodium citrate and 18.18% (w/w) PEG1000, at pH 7.0.

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KEYWORDS: tannase, Aspergillusficuum, aqueous two phase system, sodium citrate

1. INTRODUCTION

Tannin acyl hydrolase (EC 3.1.1.20), also known as tannase, is an inducible enzyme

which can be produced in the presence of inducer such as tannic acid by various

microorganisms, mainly by filamentous fungi, such as Aspergillus and Penicillium[1]

, and

also by yeast [2-4]

and bacteria [5-8]

. Tannase consists of esterase and depsidase activities

by which it can catalyze the ester and depside bonds of hydrolysable tannins to release

glucose and gallic acid[9, 10]

. As a commercially valuable enzyme, tannase has been

extensively used in a variety of fields including food, beverage, chemical and

pharmaceutical industries. In the pharmaceutical industry, tannase is mainly used in the

manufacture of gallic acid, a key intermediate required for the preparation of a

well-known antibacterial drug, trimethoprim[11]

. In addition, this enzyme is used in the

elaboration of instant tea, and acted as clarifying agent in some wines, juices of fruits and

refreshing drinks with coffee flavor.

Generally, the effective utilization of this enzyme calls for some requirements in the

purity. Purification is, therefore, a determinant step for the preparation of pure enzyme.

Several methods have been developed for the purification of tannase from a variety of

microbes [12-14]

. In most cases, the first step of tannase purification is ammonium sulfate

or acetone precipitation [15]

, which leads to a concentration of crude enzyme as well as

purification. Then column chromatography techniques such as ion-exchange

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chromatography and gel filtration chromatography are employed, which results in a high

level of purity. Nevertheless, the scale up of these methods is difficult since the use of

multistep procedures results in high loss of yield, and high costs of supplies and operation.

Thus, the development of an effective and economically advantageous method for

tannase purification is a challenging area. In this respect, aqueous two phase extraction

(ATPE) could be an attractive option due to their distinctive advantages such as mild

environment for the biomolecules, short time for partition equilibrium, ease of scale-up

and low cost. In view of this, purification of tannase has been reported recently

employing PEG/potassium phosphate system, resulting in 96% enzyme recovery with a

7.0-fold increase in purity[16]

. The major drawback of this system, however, is the

phase-forming salt used, which can lead to high phosphate concentration in the effluents,

and therefore cause environmental problems in the large-scale process. Fortunately, this

problem can be avoided by using biodegradable salts such as citrate. In fact, there has

been a tendency to use PEG/citrate systems for the recovery of biomolecules due to the

biodegradability and lower environmental toxicity of citrate compared to some other

well-studied salts, such as inorganic phosphate and sulfate salts [17-20]

.

To the author’s knowledge, there has to date been no available literatures about tannase

production by Aspergillusficuum. We therefore conducted this study to estimate the

capability of A. ficuum Gim 3.6 to produce tannase. Besides, the potential of APTE for

the partial purification of crude tannase was investigated.

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2. EXPERIMENTAL

2.1. Microorganism

Aspergillusficuum Gim 3.6 was purchased from Microbial Culture Collection Center of

Guangdong Institute of Microbiology(Guangdong, China), and maintained on potato

dextrose agar (PDA) slants at 4oC in a refrigerator.

2.2. Chemical Reagents

Propyl gallate, tannic acid and rhodanine were obtained from Tianjin Kermel Chemical

Reagents Development Center, Tianjin, China. Polyethylene glycols (PEGs) with average

molecular weight of 600, 1000, 4000 and 6000g/mol were purchased from Chengdu

Kelong Chemical Co., Ltd., Chengdu, China. All other chemical reagents were of

analytical grade.

2.3. Inoculum Preparation

The original strain was activated by scraping one loop of the strain to the PDA slant and

incubated at 30 oC for 4~5 days. For inoculum preparation, 10 ml of sterile normal saline

(0.9% NaCl) was added to the fully sporulated agar slant, and the cultures were gently

scraped with a sterile inoculating loopto dislodge the conidia off the slant. The resulting

suspension containing approximately 1.1×109

spores/ml was used as inoculum for

subsequent fermentation experiments.

2.4. Tannase Production Under Solid State Fermentation

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Solid state fermentation (SSF) was employed for the production of extracellular tannase.

Five grams of wheat bran was added to a 250 ml Erlenmeyer flask, moistened with 5.0 ml

of salt solution (containing NH4Cl 10g/L, MgSO4·7H2O 2g/L, NaCl 2 g/Land tannic acid

20 g/L, pH 5.0) and autoclaved at 121 oC for 20 min. After cooling, the solid medium was

inoculated with 1 ml (1.1×109 spores) of the spore suspension (as prepared above). The

contents were fully mixed and then incubated at 30 oC for 72 h. In this period, the solid

substrate was scattered at irregular intervals to breakup any aggregates so as to ensure the

normal growth of microbial cells and enzyme synthesis.

After the desired incubation time, the extraction of crude enzyme was done by adding 50

ml of citrate buffer (0.1M, pH5.0) to the mouldy fermented medium. The contents were

agitated in a shaker at 160 rpm for 1~2 h, filtrated through double-layer cheese cloth, and

the filtrate was centrifuged at 6790×g for 10 min. The supernatant was collected and

stored at 4oC for further analysis.

2.5. Detection Of Tannic Acid Degradation Products By Thin-Layer

Chromatography (TLC)

The tannic acid degradation by A. ficuum Gim 3.6 was detected using thin-layer

chromatography (TLC) method. The thin layer plates (10 × 10 cm) were covered with

silica gel G to a thickness of 0.5 mm, air-dried and activated in an oven at 105oC for 30

min. Solutions of gallic acid and pyrogallol standards at a concentration of 4 mg/ml were

prepared in methanol. Aliquots (5μl) of standards along with enzyme samples from 12 h,

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24 h and 48 h incubation were spotted on the silica gel plate. A mixture of chloroform,

ethyl acetate and formic acid (4:4:1) was used as the mobile phase. The chromatography

was run in an airtight container for 30 min. After drying, the plate was sprayed with

FeCl3 solution (0.01 g/ml, prepared in methanol), and then placed in an oven at 105oC for

5 min. The degradation products of tannic acid by A. ficuum Gim 3.6 appeared as colored

spots were identified by their respective Rf values.

2.6. Single-Factor Optimization Of Tannase Production

Various optimum parameters required for maximum tannase production by A. ficuum

Gim 3.6 in the solid state fermentation were determined. The effects of incubation period

(20 h~140 h), incubation temperature (25oC~50

oC), pH of salt solution (3.0~8.0) and

tannic acid concentration (1%~8%, w/v)on tannase production by A. ficuum Gim 3.6were

primarily investigated. Then, the effect of different inoculum sizes (0.2 ml~2.5 ml) on

tannase production was studied. Based on the optimum conditions determined above, the

SSF was conducted at different solid-liquid ratios (1:0.6~1:1.8) to obtain maximum

tannase production. All the experiments were performed in triplicate and average values

were reported with standard deviation.

2.7. Phase Diagrams

Phase diagrams for PEG/sodium citrate aqueous two-phase systems (ATPSs)were

obtained experimentally following the cloud point method[21]

. Firstly, a given mass of

40% (w/w) PEG stock solution was added into a 50-ml test tube. Then, a 40% (w/w)

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sodium citrate stock solution was added dropwise to the PEG solution to make the

mixture turbid, which indicated the beginning of the formation of a two-phase system.

The composition of this mixture was recorded and taken as a binodal point. A small

amount of distilled water was then added dropwise to the tube until the turbidity

disappeared. The above steps were repeated until enough points were obtained to form a

binodal curve.

2.8. Aqueous Two-Phase Extraction

Aqueous two-phase systems were prepared (based on the phase diagrams) in 10-ml

graduated centrifuge tubes by the addition of appropriate amounts of PEG (MW 600,

1000, 4000, and 6000) stock solution, sodium citrate stock solution, and enzyme extract.

Distilled water was added whenever necessary to make the total mass of 10 g. The pH of

the system was assumed to be equal to that of the sodium citrate stock solution. The pH

of the sodium citrate stock solution was adjusted with 0.1M HCl solution. The mixture

was thoroughly mixed in a vortex mixer for about 5 min and centrifuged at 954×gat room

temperature for 10 min to speed up phase separation. The volume of each phase was

directly estimated in graduated centrifuge tubes and aliquots from each phase were taken

for the activity assay and protein determination. All the tests were performed in duplicate

and average values were reported.

Effects of various parameters including phase-forming salt, molecular weight of PEG, pH

and stability ratio on tannase partition and purification in aqueous two-phase system

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(ATPS) were studied. The stability ratio, previously reported by Zhou et al. [22]

, was

selected as an influential factor on tannase portioning, as it is similar to the tie line length

(TLL) which, in most cases, is one of the studied parameters in ATPE experiments.

Moreover, determination of stability ratio is relatively simple. The stability ratio is

illustrated in Figure 1. In the figure, point A is the point on the binodal curve, and point B,

C, D, and E are the operating points chosen for the ATPE experiments. The

corresponding value of stability ratio of point B was calculated according to equation (1),

and the stability ratio values of other three points can be calculated in the same way.

OB

ABratio Stability

(1)

Where AB and OB represent the length of the segments from A to B, and O to B,

respectively.

Following this calculation method, the stability ratio values of B, C, D, and E were

obtained as 0.0625, 0.1176, 0.1667 and 0.2500, respectively.

2.9. Estimation Of Partition Coefficient, Activity Recovery And Purification Fold

The partition coefficients of tannase (Ke) or total proteins (Kp) were defined as the

enzyme activity or protein concentration in the top phase to that in the bottom phase, as

shown in equations (2) and (3):

b

te

A

AK

(2)

b

tp

C

CK

(3)

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Where At and Ab are the tannase activity in the top and bottom phases, respectively. Ct and

Cb are the equilibrium concentrations of the protein in the top phase and bottom phase,

respectively.

Activity recovery (Y) was defined as the total activity in the top phase to that in the initial

added crude enzyme and can be calculated by equation (4):

%100(%)ii

tt

VA

VAY

(4)

Where Vt and Vi are the volumes of the top phase and initial crude enzyme extract,

respectively. Ai is the enzyme activity in the crude extract.

Purification fold (PF) was defined as the ratio of the specific activity in the top phase to

that in the crude enzyme and can be calculated according to equation (5):

ii

tt

CA

CAPF

(5)

Where Ci is the protein concentration in the initial crude enzyme.

2.10. Determination Of Enzyme Activity And Protein Concentration

Tannase activity was determined as per the rhodanine method of Sharma et al. [23]

with a

slight modification, using propyl gallate (PG) instead of methyl gallate as the substrate.

In this method, tannase catalyzes the hydrolysis of the galloyl ester linkage of the

substrate PG to release gallic acid which reacts subsequently with rhodanine to give a red

complex with a maximum absorbance at 520 nm.The substrate solution (10 mM PG

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prepared in 0.1 M citrate buffer, pH5.0) and enzyme sample were preincubated at 30oC

for 10 min, and the enzyme reaction then started. For simplicity, we diagramed the assay

procedure of tannase activity in Figure 2. One unit of tannase was defined as the amount

of enzyme required to release 1μmol of gallic acid per minute under the assay conditions.

Tannase activity is expressed as units per gram of dry substrate (U/gds).

Protein concentration was determined according to the method proposed by Bradford [24]

using bovine serum albumin as the standard. The absorbance was measured at 595 nm

using a UV-vis spectrophotometer (Shimadzu UVmini-1240, Kyoto, Japan). To avoid the

interference of phase components on protein determination, samples from equilibrium

phases were diluted and analyzed against blanks with the same composition but without

enzyme extract.

3. RESULTS AND DISCUSSION

3.1. Tannase Production Under Solid State Fermentation

Tannase can be obtained from microbial, animal and vegetal sources, but microorganisms

(especially filamentous fungi) are commonly used for commercial production since

tannase obtained by this way is more stable [25]

. Although microbial production of tannase

from Aspergillus species has been well documented, the enzyme productivity has been

relatively low, such as 4.36 U/gds[26]

and 3.42 U/gds[27]

for Aspergillusoryzae, and 6.44

U/gds for Aspergillusniger ATCC 16620 [28]

. In this work, tannase production using a new

fungal strain, A. ficcum Gim 3.6, was attempted. After the specific period of incubation,

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crude enzyme was extracted and qualitative analysis of the tannic acid degradation

products and quantitative assay of tannase activity were carried out. Thethin layer

chromatographic separation of tannic acid degradation products is illustrated Figure 3(A).

When compared with standards, the spots of gallic acid and pyrogallol were detected in

the TLC plates. The appearance of gallic acid and pyrogallol demonstrates that tannic

acid was degraded byA. ficcum Gim 3.6 during the fermentation process. It has been

known that tannase can specifically hydrolyze the galloyl ester bonds of tannins.

According to the result of TLC, we hypothesised that tannic acid in the culture medium

was degraded by A. ficcum Gim 3.6 (by the secretion of tannase) to gallic acid in the early

stage of fermentation, thereafter gallic acid decarboxylase catalyze the second step in the

degradation of the tannic acid, the decarboxylation of gallic acid to pyrogallol. The

proposed biochemical pathway for the degradation of tannic acid by A. ficcum Gim 3.6 is

presented in Figure 3(B). Quantitative determination of tannase indicated that the crude

extract shown an activity of 8.65 U/gds. From the above results it could be unequivocally

deduced that A. ficcum Gim 3.6 possesses the metabolic ability to degrade tannic acid by

secreting the enzyme tannase.

3.2. Single-Factor Optimization Of Tannase Production

Single-factor tests were carried out to evaluate the effects of various process parameters

on tannase production, and the results are presented in Figure 4. The effect of incubation

period and incubation temperature on tannase production is shown in Figure 4(A). It was

observed that the enzyme production increased progressively from 20 h to 60 h, and an

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incubation period of 60 h was best for maximum tannase production (9.94 U/gds);

thereafter, the production of tannase started decreasing with time, which might be due to

the inhibition effect of metabolites produced by microbes [29]

. Moreover, with the

prolonging of time, substrate scarcity would occur, and the secretion of some toxic

substances could lead to cell lysis.

A significant difference in the enzyme production was observed when the fermentation

was performed at different temperatures (25~50oC). Among the various incubation

temperatures, the maximum enzyme yield of 9.34 U/gds was obtained at 30oC, whereas

above 30oC, the enzyme activity decreased (Figure 4(A)). The depletion of enzyme

production at higher temperature might be due to the mycelial growth inhibition caused

by the sporulation induced with increased temperature [30]

. In addition, a high temperature

may lead to evaporation of liquid content which is required for microbial growth and

enzyme synthesis.

The influence of initial pH of salt solution on the production of tannase is presented in

Figure 4(B), from which it can be seen that an initial pH of 5.0 gave maximum tannase

production (9.23U/gds). Below or above 5.0, there was a decrease in tannase activity.

This is in good accordance with some other reports [30-32]

. It is generally known that the

metabolic activities of microorganisms are very sensitive to changes in pH, microbial

growth and tannase secretion would be inhibited in the unsuitable pH circumstance.

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A certain level of tannic acid is needed for tannase production since tannase is an

inducible enzyme. The enzyme production increased with increasing inducer

concentration (ranging from 1~8%) up to 2.0 % (w/v) beyond which a declining trend of

tannase activity was observed (Figure 4(B)). This is because tannic acid itself is toxic for

the microbes which may retard their growth [33]

.

Inoculum size in the studied range had significant impact on tannase production. From

Figure 4(C), it was found that an inoculum size of 1.0 ml gave the maximum tannase

yield, when the content of wheat bran was 5 g. A smaller or larger volume of inoculum

led to decrease in tannase synthesis as it may be insufficient for complete use of the

available substrate to the mycelial growth and enzyme synthesis when a small volume of

inoculum was used [28, 34]

, and increases in the depletion of nutrients may occur when the

inoculum size was above 1.0 ml[35]

.

As for the effect of solid-liquid ratio on tannase production, it can be seen from Figure

4(C) that the maximum amount of tannase production (12.98U/gds) was attained when

the ratio of wheat bran to salt solution was 1:1. With a high level of solid content, the

liquid volume may not be sufficient for complete leaching of the product from the

fermented biomass, resulting in a decrease of enzyme activity. Likewise, the enzyme

activity may decrease with a high volume of liquid, as the oxygen supply for microbial

growth in the culture medium may decline in such condition[30]

.

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3.3. Aqueous Two-Phase Extraction

3.3.1. Phase Diagrams

In order to predict the volume and composition of the two phases, phase diagrams for

PEG/sodium citrate ATPSs with different PEG molecular weight (MW 600, 1000, 4000

and 6000g/mol) were constructed, as shown in Figure 5. It is evident from the figure that

an increase in the PEG molecular weight led to lower PEG concentrations required for

phase separation, and binodal curves became more asymmetric and close to the origin.

We inferred that this may be caused by the increase in the incompatibility between the

system components due to the more hydrophobic character of PEGs of higher molecular

weight.

3.4.2. Selection Of System Type And Phase-Forming Salt

In general, there are two major types of ATPS, namely polymer/polymer system and

polymer/salt system. ATPSs composed of a polymer (e.g. PEG) and a salt (e.g. phosphate)

have been widely used and are an attractive alternative to polymer/polymer systems due

to the use of inexpensive phase components [36]

. Additionally, polymer/salt systems have

a relatively lower phase viscosity, which enhances the segregation of the two phases,

meaning that time for phase equilibrium can be saved. These advantages of polymer/salt

systems over polymer/polymer systems could be highly advantageous in industrial

processes [37]

. For this reason, polymer/salt system was selected in our work for tannase

purification.

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After selecting the type of system, we attempted to find out the most suitable salt for

partitioning of the enzyme. The effects of different salts on enzyme activity recovery

were studied using four different salts, namely ammonium sulphate, magnesium sulphate,

dipotassium phosphate and sodium citrate. In this step, crude enzyme extract was mixed

with the same volume of 30% (w/w) salt solution, kept at room temperature for

approximately 20 min, and then analyzed for enzyme activity. The results are shown in

Table 1. Compared with other salts, sodium citrate exhibited good compatibility for the

enzyme, as is evident from the highest recovery of activity (92.36%). Though magnesium

sulphate has been frequently used as the phase-forming salt of ATPS, it was found to be

unsuitable in this study because of the turbidity formed in the activity assay process,

probably due to the reaction of MgSO4 with KOH, forming the insoluble compound, Mg

(OH)2. Hence, sodium citrate was selected as phase-forming salt for further studies.

3.3.3. Effect Of PEG Molecular Weight

In order to select the most suitable molecular weight of PEG for purification of tannase,

various ATPSs having PEG of different molecular weights (MW 600, 1000, 4000, and

6000) and sodium citrate were employed for partitioning experiments. Other parameters

such as total phase composition, temperature and pH of all the systems were kept

constant. The results are presented in Figure 6 and Table 2. In all the systems, Ke values

were above 1.0, which means that the target enzyme was preferentially partitioned to the

top phase. This is dissimilar to the finding of Rodríguez-Durán et al. [16]

who found that

tannase was partially purified in the bottom phase. Notwithstanding, the target enzyme

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partitions to the top PEG-rich phase represents a technical advantage, as it is possible to

recover the polymer from the system by inducing the formation of a new bottom phase,

offering to the biomolecule a new possibility of migration, which assure the low-cost of

the purification process [38]

. It should be noted that the behavior of a particular

biomolecule in ATPS is rather unique since the chemical and physical interactions

involved in the partitioning process are very complex.

The partition coefficients of enzyme (Ke) and total protein (Kp) were found to decrease

with an increase in molecular weight of the PEG (Figure 6). It can be observed from

Table 2 that the enzyme activity recovery as well as purification fold of systems with

PEG 600 and 1000 was better than the systems with higher PEG molecular weight (PEG

4000 and 6000). These observations could be interpreted in terms of excluded volume

effect and hydrophobicity of the PEG molecule. With an increase in the molecular weight

of the PEG, the proteins to be partitioned in the system were subjected to a stronger

excluded volume effect imposed by the high molecular PEG. In other words, the free

volume in the PEG-rich phase (top-phase) significantly decreases with the increase in

PEG molecular weight. As a result, most of the biomolecules in the system selectively

partitioned to the bottom phase. On the other hand, as the molecular weight of the PEG

increases, the hydrophobic groups/hydrophobic area ratio decreases, resulting in an

increase in hydrophobicity [39]

. Consequently, the target enzyme as well as contaminating

proteins partitioned to the bottom phase.

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Table 2 shows that activity recovery of the systems with PEG 600 and PEG 1000 were

close; however, the latter was considered as the better one since the purification fold

(2.07-fold) of which was higher. The incommensurate tendency of the Ke and Kp of the

two systems (Figure 6) indicates that the PEG with low molecular weight is of poor

selectivity (S=Ke /Kp). Hence, PEG 1000 was selected for further studies.

3.3.4. Effect Of Ph

In order to evaluate the effect of pH on partition and purification, experiments were

carried out using the PEG1000/sodium citrate (20%/20%, w/w) system at different pH

values. The range of pH of the ATPSs was chosen as 3.0~8.0 since the pH stability of

tannase is around 3.5~8.0 [40]

. The results are shown in Table 3. When the partitioning

experiments were performed at pH 3.0 and 4.0, no phase separation was observed. This

finding illustrates that a higher concentration of phase-forming components is needed for

phase separation in the systems at low pH value. This interpretation can also be affirmed

by the finding of Marcos et al. [21]

who found that although the shape of the binodal lines

was similar for systems at different pH values, the tie line length for systems with the

same composition increased with increasing pH. From Table 3, the highest tannase

activity recovery (78.41%) and purification fold (1.92-fold) were attained at pH 7.0.

Hence, the pH value of 7.0 was selected for further studies.

3.3.5. Effect Of Different Stability Ratios

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To identify the most suitable stability ratio for tannase purification, experiments were

performed using PEG1000/sodium citrate systems of different stability ratios, at pH 7.0.

The results are shown in Table 4. An increase in the specific activity was observed as

stability ratio increased from 0.0625 to 0.1176, and the stability ratio of 0.1176 gave

maximum activity recovery (77.17%) with a purification fold of 2.74. Further increase in

stability ratio from 0.1176 to 0.2500 resulted in decreases in activity recovery and

specific activity. The excluded volume theory shows that increase in the PEG

concentration or its molecular weight induces a decrease of the protein solubility in the

phase where the protein is located. With the increase in the stability ratio, the

concentration of PEG is increased, which led to the increase of excluded volume effect.

Thus, the activity recovery as well as purification fold was lowered with further increase

in the stability ratio above 0.1176.

4. CONCLUSIONS

A. ficuum Gim 3.6 has been proved to possess metabolic ability to degrade tannic acid

and produce a relatively high yield of extracellular tannase under the solid state

fermentation. The production of tannase reached maximum at 60 h of incubation and

addition of 2.0% (w/v) tannic acid as inducer, 1 ml of inoculum size and 1:1 of

solid-liquid ratio at temperature of 30oC and pH of 5.0.

The partial purification of crude tannase in ATPS was attempted. PEG1000/sodium citrate

system was found to be the most suitable system. The best result of purification

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(2.74-fold) with an activity recovery of 77.17 % was obtained in the PEG1000/sodium

citrate system having 17% (w/w) of sodium citrate and 18.18% (w/w) of PEG 1000, at

pH 7.0. These results demonstrate that ATPE could be an effective technique for the

partial purification of tannase.

ACKNOWLEDGMENT

This work was supported by Guangxi Natural Science Foundation

(2010GXNSFC013004).

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TABLE 1 Activity recovery of tannase using different salts

Salt Enzyme activity recovery (%) pH

Ammonium sulphate 47.66 5.0

Magnesium sulphate _* 5.8

Dipotassium phosphate 83.18 7.2

Sodium citrate 92.36 7.8

*Activity could not be determined due to the turbidity formation after the addition of

KOH solution to the reaction mixture.

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TABLE 2 Effect of PEG molecular weight on tannase partitioning

PEG

MW

Total activity

(U)

Total

protein

(mg)

Specific

activity

(U/mg)

Activity

recovery (%)

Purification

fold

600 1.03 0.19 5.42 76.30 1.48

1000 0.98 0.13 7.54 72.59 2.07

4000 0.86 0.18 4.78 63.70 1.31

6000 0.77 0.20 3.85 57.04 1.05

Crude 1.35 0.37 3.65 100 1

Phase system: PEG/sodium citrate, 16%/20% (w/w), at 23 oC, pH 7.8.

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TABLE 3 Effect of pH on tannase partitioning

pH Total

activity

(U)

Total

protein

(mg)

Specific

activity

(U/mg)

Activity

recovery

(%)

Purification

fold

5.0 0.61 0.21 2.90 69.32 0.73

6.0 0.65 0.09 7.22 73.86 1.81

7.0 0.69 0.09 7.67 78.41 1.92

8.0 0.60 0.08 7.50 68.18 1.88

Crude 0.88 0.22 4.00 100 1

Phase system: PEG1000/sodium citrate, 20%/20% (w/w), at 23 oC.

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TABLE 4 Effect of different stability ratios on tannase partitioning

Phase

composition

(w/w, %)

Run Stability

ratio

PEG Sodium

citrate

Total

activit

y (U)

Total

protei

n

(mg)

Specifi

c

activity

(U/mg)

Activity

recover

y (%)

Purificatio

n fold

B 0.0625 16 17.11 0.88 0.09 9.78 69.29 2.46

C 0.1176 17 18.18 0.98 0.09 10.89 77.17 2.74

D 0.1667 18 19.25 0.97 0.12 8.08 76.38 2.04

E 0.2500 20 21.39 0.95 0.16 5.94 74.80 1.50

Crude 1.27 0.32 3.97 100 1

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FIGURE 1 Illustration of stability ratio in PEG1000/sodium citrate ATPS. O: original

point; A: point on the binodal curve; B, C, D, and E: selected ATPS points. The stability

ratio values of B, C, D, and E are 0.0625, 0.1176, 0.1667 and 0.2500, respectively. The

corresponding compositions of these operating points are B (16, 17.11), C (17, 18.18), D

(18, 19.25), and E (20, 21.39), respectively. The abscissa and ordinate represent the

concentration (w/w) of sodium citrate and PEG1000 in the APTS, respectively.

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FIGURE 2 Schematic diagram of tannase assay method according to Sharma et al. [23].

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FIGURE 3 (A) TCL profile of degradation of tannic acid by A. ficcum Gim 3.6.

GA-galic acid, PA- pyrogallol; (B) Proposed pathway for tannic acid degradation by A.

ficcum Gim 3.6.

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FIGURE 4 Effects of various parameters on tannase production. (A) Incubation period (h)

and incubation temperature (oC), (B) Initial pH and tannic acid concentration (%, w/v),

(C) Inoculum size (ml) and solid-liquid ratio.

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FIGURE 5 Phase diagrams for PEG/sodium citrate ATPSs with different PEG molecular

weight.

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FIGURE 6 Effect of PEG molecular weight on partition coefficient of tannase (Ke) and

total protein (Kp).

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