Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e8, 2014 www.elsevier.com/locate/jbiosc
Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract Joo Hyun Baik,1, z Hyung Joo Suh,2, 3, z So Young Cho,2 Yooheon Park,2 and Hyeon-Son Choi2, * Cosmax Bio INC, Jecheon 390-250, Republic of Korea,1 Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea,2 and Department of Public Health Science, Graduate School, Korea University, Seoul 136-7033, Republic of Korea3 Received 11 February 2014; accepted 16 April 2014 Available online xxx
Tannases are important enzymes in the antioxidant potential of tea leaves. In this study, we evaluated the effect of two tannases (T1 and T2) on biotransformation of tea polyphenols and antioxidative activities from catechins in green tea extract (GTE). The T1 tannase-catalyzed reaction was inhibited by the addition of >2.0% GTE substrate, whereas the T2catalyzed reaction was not inhibited, even by addition of 5.0% GTE. Furthermore, the T1 tannase-catalyzed reaction was inhibited by addition of 10 mg mLL1 EGCG, whereas the T2 tannase-catalyzed reaction did not display any inhibitory effect. These results indicate that T2 tannase was more tolerant than T1 tannase to substrate inhibition in degallation reactions. Specifically, the substrate EGCG (90,687.1 mg mLL1) was transformed into gallic acid (50,242.9 mg mLL1) and EGC (92,598.3 mg mLL1) after 1-h treatment with T2 tannase (500 U gL1). The tannase-mediated product displayed higher in vitro radical-scavenging activity than the control. IC50 value of GTE on ABTS and DPPH radicals (46.1 mg mLL1 and 18.4 mg mLL1, respectively) decreased markedly after T2 tannase treatment (to 35.8 mg mLL1 and 15.1 mg mLL1, respectively). These results indicate that T2 tannase treatment of GTE enhanced its radical-scavenging activity, an increase that was also observed in the reaction using EGCG substrate. Taken together, our results revealed that T2 tannase is more suitable for biotransformation of catechins in GTE than T1 tannase, and T2 treatment provides an enhanced radical-scavenging effect. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Tannase; Biotransformation; Tea catechins; Antioxidative activity; Substrate inhibition]
Tea polyphenols constitute one of several essential components of tea that affect physiological functions. Catechins are the primary polyphenols found in tea, accounting for 75e80% of its soluble ingredients (1), including epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC), gallocatechin (GC) and catechin (C). EGCG constitutes over 50% of catechin content in tea (2). Tannase or tannin acyl hydrolase (EC, 3.1.1.20) catalyzes the hydrolysis of ester and depside bonds present in hydrolyzable tannins or gallic acid esters, such as EGCG or ECG, thereby releasing glucose or gallic acid as products (3). The release of gallic acid is physiologically beneficial due to its significant antioxidant potency (4). We previously reported that increase of gallic acid levels by tannase treatment was associated with an increase in antioxidant activity (5). Gallic acid is used in the synthesis of propyl gallate, which is an antioxidant primarily found in fats and oils, as well as in beverages. Tannase is also extensively used in the preparation of instant tea, acorn wine, and coffee-flavored soft drinks; the clarification of beer and fruit juices; and the detannification of foods (3,6,7). Many studies of tannase-mediated biotransformation of tea catechins have been performed (5,8,9). Our previous study (10)
* Corresponding author. Tel.: þ82 2 940 2764; fax: þ82 2 940 2859. E-mail address: [email protected] (H.-S. Choi). z The first two authors contributed equally to this work.
reported that tea catechins (such as EGCG or ECG) were hydrolyzed by tannase action (to generate EGC or EC, respectively, as well as gallic acid) by using >1% tea extract as a substrate. However, biotransformation of catechins by tannase was observed to be affected by substrate-mediated dose-dependent inhibition (5). Albertse (11) also reported the inhibition of tannase at higher concentrations of EGCG. Tea polyphenols, such as EGCG, are known to be involved in enzyme inhibition as well as the formation of precipitants in tea drinks (12,13). Substrate-mediated inhibition has been an important limitation on the widespread use of tannases. Therefore, the discovery of a tannase that is not susceptible to substrate inhibition is an important goal for the use of tannases in the biotransformation of tea catechins. At the present, many companies manufacture tannases of varying catalytic properties (7) using multiple methods. Tannases from Kikkoman (Japan) and Biocon (India) are manufactured by solid-state fermentation (SSF). The reactivity of tannase on substrates can depend on the enzyme source, culture methods, degree of purification, etc. (7). In general, tannases have been isolated from microbial sources, such as fungi, using SSF or submerged fermentation (SmF). Several differences in tannase production from microbes have been observed between SSF and SmF methods (14,15). For example, substrate concentrations are recognized to affect the regulatory mechanisms of tannase production (14,15). In this study, we examined the effect of two fungi-derived tannases from different manufacturers on biotransformation of tea
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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catechins and substrate inhibition. The antioxidative effects of tannase hydrolysates between the two types of tannases were also compared. This study shows that T2 tannase treatment leads to vastly improved biotransformation of tea catechins over T1 tannase treatment, without concomitant substrate inhibition compared. MATERIALS AND METHODS Materials and chemicals Green tea extract (GTE) was purchased from Zhejiang Huang Minghuang Natural Products Development Co., Ltd. (Beijing, China). The extract was determined by high-performance liquid chromatography (HPLC) to be composed of 71.9% catechins, including 40.9% EGCG. Acetic acid and acetonitrile (HPLC analytical grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). HPLC-grade standard gallic acid (GA), EGC, EC, EGCG, ECG, and standards were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). The remaining reagents used were of analytical grade. Tannases were purchased from Kikkoman Corporation (Tokyo, Japan) and Bision Biochem. Co. (Sungnam, Republic of Korea). Tannase treatment and determination of catechins by HPLC Thirty mg of tannase (500 U g1) was added to 50 mL of 1% GTE solution, and the reactions were performed at 35 C. The tannase reactant of green tea was obtained at regular intervals until the completion of hydrolysis (10). The inactivation of hydrolysis was performed by heating the sample at 100 C for 10 min after reaction. The content of each catechin was measured using an HPLC system equipped with a hypersil C18 column (5 mm, 25 0.46 cm ID) and a UVeVIS detector. The mobile phase contained 1% acetic acid (solvent A) and acetonitrile (solvent B) with a linear gradient starting at an A/B ratio of 92/8 and completing at 73/27 over a 40-min period at a flow rate of 1 mL min1. Identification of individual catechins and GA was based on a comparison of the retention times of the sample peaks with those of authentic reference standards. The quantity of each constituent in green tea was estimated from the integrated data (16). Inhibitory activity of EGCG and gallic acid The effect of EGCG and gallic acid addition was tested with GTE. Gallic acid (5 and 10 mg mL1) and EGCG (1, 2, 5 and 10 mg mL1) were added to the enzymatic reaction mixture (50 mL) of tannase. Thirty mg of tannase (500 U g1) was added to 50 mL of 1% GTE solution, and the reactions were performed at 35 C for 2 h. The inactivation of hydrolysis was performed by heating at 100 C for 10 min after reaction. Samples were removed at regular intervals until the completion of hydrolysis and assayed to determine changes in the level of catechins.
Scaleup for the biotransformation of GTE Six gram of tannase was added to 10 L of 10% GTE solution, with the reactions performed at 35 C. The tannase reactant of green tea was obtained at regular intervals until the completion of hydrolysis. The inactivation of hydrolysis was performed by heating at 100 C for 10 min after the reaction. The reactant was lyophilized and used for the assay of catechin and radical-scavenging activity. Antioxidant activities of green tea extract The scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured, according to the method of Cheung et al. (17) The scavenging activity of the 2,2-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical was measured according to the method of Re et al. (18) The DPPH and ABTS radical-scavenging activities were calculated as follows: Radical scavenging activityð%Þ ¼
. 1 Asample Acontrol 100
(1)
where Asample is the absorbance in the presence of sample, and Acontrol is the absorbance in the absence of sample. The half-maximal inhibitory concentration (IC50) value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50%. Statistical analysis All expressed values are mean values of triplicate determinations. All statistical analyses were performed using the Statistical Package for Social Sciences version 12.0 (SPSS, Chicago, IL, USA). Differences among the samples were evaluated statistically by one-way analysis of variance (ANOVA) and Tukey’s New Multiple Test. All data were two-sided at a 5% significance level, and have been reported as mean deviation (SD).
RESULTS Effect of GTE substrate concentration on tannase T1 and T2 are Aspergillus niger-derived tannases produced using different methods. T1 tannase obtained from Kikkoman was produced by SSF, while T2 tannase from Bision was produced by SmF. To measure the effect of substrate concentration in tannase T1- and T2mediated reactions, several concentrations of green tea extract (0.5e5.0%) were added to the reaction mixture, followed by HPLC analysis of their products (Fig. 1). The addition of 0.5% GTE resulted in an increase of the quantity of gallic acid as a product of the T1
FIG. 1. Effect of GTE concentration on tannase T1 (A, B) and T2 (C, D) activity. The reactions were performed at 35 C and pH 5.0 at various GTE concentrations.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
VOL. xx, 2014 and T2 tannase-mediated reaction after a 1-h incubation period. The level of gallic acid generated from the T2 tannase-mediated reaction was slightly higher than that of the T1 tannase-mediated reaction with 0.5% GTE; T1 tannase generated 1600 mg mL1 gallic acid, while T2 tannase generated 1800 mg mL1 gallic acids after a 1-hr incubation period (Fig. 1). Conversely, the quantity of EGCG, a substrate of the T1 and T2 tannase reaction, decreased to negligible levels after the 1-h reaction period, indicating that almost all of the EGCG was utilized for enzyme-catalyzed conversion into gallic acid. When 1.0% GTE was added, the T1 enzyme reaction proceeded at a slower rate than the T2 enzyme reaction. In the T2 reaction, gallic acid production was achieved up to a gallic acid concentration of 3000 mg mL1 within 1 h, whereas the T1 tannase reaction required 3 h to generate gallic acid to a concentration of 3000 mg mL1. Furthermore, gallic acid production by the T1 reaction was greatly decreased in the treatment of >2.0% GTE, suggesting that the T1 tannase reaction was subject to inhibition at these substrate levels. T2 tannase generated gallic acid concentrations of 6000 mg mL1 in a 1-h period, consuming all quantities of EGCG within 30 min, suggesting that T2 tannase was not subject to substrate inhibition at 2% GTE, displaying a higher enzymatic activity in the same substrate than T1. Even the addition of 5% GTE did not inhibit T2 tannase-catalyzed gallic acid production, whereby all quantities of EGCG in GTE were consumed within 2 h. Thus, T2 tannase led to a larger quantity of product in a shorter reaction time compared with T1 tannase. Our data indicated that SmF-derived T2 tannase has a much greater capacity for conversion of EGCG into gallic acid than SSF-derived T1 tannase. Effect of EGCG concentration as substrate on tannase Fresh green tea leaves typically contain 36% polyphenols, primarily catechins. EGCG is the predominant catechin
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present in green tea leaves (48e55% of total polyphenols) (19). The substrate GTE is composed of 71.9% catechins, including 40.9% EGCG, which may be responsible for the inhibitory effect of GTE in the tannase reaction. Therefore, we examined the effect of EGCG concentrations as a tannase inhibitor on both tannase T1 and T2 reactions (Fig. 2). The addition of 1 and 2 mg mL1 EGCG did not inhibit T1 and T2 tannase reactions in which gallic acid product levels were measured at 500 mg mL1 and 1000 mg mL1, respectively. Additionally, 1 and 2 mg mL1 of added EGCGs were completely consumed by 10 min in both tannase reactions. This result demonstrated that most EGCG was consumed for conversion into gallic acid within the 30-min reaction period catalyzed by T1 and T2 tannase (Fig. 2). Addition of 5 mg mL1 EGCG did not inhibit T1 or T2 tannases, but displayed the difference in rate of product generation between the two tannases. T1 tannase activity generated 2000 mg mL1 of gallic acid from 5 mg mL1 of EGCG within a 1-h period, while T2 tannase activity generated 2400 mg mL1 gallic acid within 10 min. In addition, EGCG levels in the T1 tannase reaction were decreased in a 2-h period, whereas the T2 tannase reaction induced a dramatic reduction of EGCG levels within 10 min. These results demonstrated that the enzymatic activity of T2 tannase was much greater than that of T1, consistent with the above GTE data (Fig. 1). Specifically, T1 tannase was completely inhibited by the addition of 10 mg mL1 EGCG without any concomitant consumption of substrate, whereas T2 tannase consumed 10 mg mL1 EGCG within 10 min to generate high levels (5000 mg mL1) of gallic acids. Our data indicated that T2 tannase apparently has stronger enzymatic capabilities than T1 tannase to convert EGCG into gallic acid, and is much more tolerant to EGCG inhibition.
FIG. 2. Effect of EGCG concentration on tannase T1 (A, B) and T2 (C, D) activity. The reactions were performed at 35 C and pH 5.0 at various EGCG concentrations in 50 mL of total volume.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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Effect of gallic acid concentration as product on tannase Gallic acid is reported to be associated with end-product repression of tannase (20). We subsequently examined the inhibitory effect of gallic acid addition on T1 and T2 tannases. When 5 and 10 mg mL1 of gallic acid was added to the reactions, gallic acid product levels increased, while EGCG levels decreased, in a 2-h reaction period compared with 0 time (control; Fig. 3). Reactions containing 5 and 10 mg mL1 gallic acid additions increased gallic acid levels by 120% and 73%, respectively. The increase width of gallic acid product in these reactions was lower than untreated control, due to the higher initial levels of gallic acid present after addition. However, three reaction groups (0, 5, and 10 mg mL1 of gallic acid) displayed similar levels of consumed EGCG during the reactions (Fig. 3), a trend that was observed in both tannases. These results demonstrated that the tannases T1 and T2 were not inhibited by the addition of gallic acids (5 and 10 mg mL1). Our data suggested that addition of gallic acid did not induce the endproduct repression of T1 and T2 tannases. Moreover, the addition of gallic acid also reduced the pH of the sample from 6.0 to 3.8 (data was not shown), although the decease of pH did not influence the T1 and T2 reaction. EGCG biotransformation by T2 tannase and radicalscavenging activity As the most abundant polyphenol in green tea, EGCG is one of the most interesting components of tea leaf, exhibiting potent antioxidant activity in vitro and in vivo (21). Additionally, tea polyphenols such as catechins (including EGCG) and gallic acid have also been considered primary effectors in the beneficial effects of tea on the human health. However, EGCG has been reported to present potential risks, including DNA damage, and is a primary ingredient providing an astringent taste to tea drink.
J. BIOSCI. BIOENG., Therefore, we transformed EGCG into degallated catechins with the catalytic action of T2 tannase and examined the antioxidative effects of resulting EGCG hydrolysates. EGCG and its hydrolysates, gallic acid and EGC, were analyzed with HPLC (Fig. 4). EGCG (90,687.1 mg mL1) was completely transformed into EGC (92,598.3 mg mL1) and gallic acid (50,242.9 mg mL1) after T2 tannase treatment for 1 h, as observed in the post-reaction HPLC chromatogram (Fig. 4). Next, we examined the free-radical scavenging activities between before and after T2 tannase reaction using the DPPH and ABTS method. Radical-scavenging capacity was proportionally increased with EGC and gallic acid contents in the bioconversion reaction. Scavenging capabilities on DPPH and ABTS radicals were also significantly enhanced after T2 tannase treatment (Fig. 5); IC50 values on DPPH and ABTS radicals after T2 treatment (14.0 and 25.5 mg mL1, respectively) were significantly lower than those before T2 treatment (17.8 and 39.6 mg mL1, respectively; P < 0.05). This result demonstrated that T2 tannase treatment increased radical-scavenging activity of the EGCG solution via enzymatic biotransformation. Increased levels of EGC and gallic acids were suspected to be responsible for the enhancement of radical-scavenging activity. Our data indicated that tannase-derived bioconversion could improve the biological function of green tea extracts. Scale-up for the biotransformation of GTE and radicalscavenging activity of biotransformed GTE We examined the availability of T2 tannase for industrial bioconversion of GTE. Total reaction volume was scaled up to 10 L, with increased T2 tannase and substrate GTE. Ten percent GTE solution was treated with T2 tannase for a 4-h period. T2 tannase treatment resulted in an increase in the amount of gallic acid product of the tannase reaction from 2480.0 mg mL1 to 39,797.5 mg mL1 after a 2-h reaction period (Fig. 6A). Moreover, the amount of EGCG substrate of the tannase
FIG. 3. Effect of gallic acid concentration on tannase T1 (A, B) and T2 (C, D) activity. The reactions were performed at 35 C and pH 5.0 at various gallic acid concentrations.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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FIG. 4. EGCG (A) biotransformation to EGC and gallic acid (B) by tannase T2. The reactions (50 mL) were performed at 35 C and pH 5.0 with EGCG (10 mg) as a substrate and tannase T2 (30 mg). Catechin was measured using an HPLC system equipped with a hypersil C18 column (5 mm, 25 0.46 cm ID) and a UVeVIS detector. The mobile phase consisted of 1% acetic acid (solvent A) and acetonitrile (solvent B) with a linear gradient starting at an A/B ratio of 92/8 and terminating at 73/27 over a 40-min period at a flow rate of 1 mL min1.
reaction decreased from 39,661.5 mg mL1 to 6.5 mg mL1 after a 2-h reaction period. Gallic acid production sharply increased with T2 treatment until 1 h and then slightly increased thereafter. The increasing trend of gallic acid was precisely associated with the downward trend of EGCG (Fig. 6A). After 2 h of T2 tannase treatment, the sample displayed a decrease in gallated catechin levels, including EGCG, ECG, and GCG, with a concomitant increase in degallated catechin levels including EGC, EC, and GC. This result demonstrated that most EGCG, ECG, and GCG were converted to EGC, EC, and GC, respectively, after a 2h reaction period (Fig. 6B). At 2 h, EGC, EC, and GC content increased from 15,149.2, 5696.7, and 2065.4 mg mL1 to 53,341.8, 14,576.9, and 3638.8 mg mL1, respectively. Our data indicated that degallation of T2 tannase functioned normally for biotransformation of catechins in a scaled-up system. The radical-scavenging activity of GTE was determined before and after T2 tannase treatment in a scaled-up system. As shown in
Fig. 7, the IC50 value of GTE for DPPH radical-scavenging was decreased from 18.4 mg mL1 to 15.1 mg mL1 by T2 tannase treatment. In addition, the IC50 of GTE on ABTS radicals was decreased to 35.8 g mL1 from 46.1 g mL1 by T2 tannase treatment. These data demonstrated that T2 tannase treatment improved the antioxidative effects of GTE via biotransformation of catechins, consistent with above data reporting a higher antioxidative activity with T2 tannase treatment in EGCG solution (Fig. 5). Our results suggested that T2 tannase may play an important role in the conversion of catechins in an industrial setting.
DISCUSSION Tea polyphenols play an important role in protein precipitation and enzyme inhibition through the formation of a variety of complexes with tea ingredients (12,13). It is known that most
FIG. 5. Effect of T2 tannase treatment on DPPH (A) and ABTS (B) radical-scavenging activity of EGCG. The half-maximal inhibitory concentration (IC50) value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50%.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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FIG. 6. GTE biotransformation by T2 tannase in scale-up system. Six gram of tannase (500 U g1) was added to 10 L of 10% GTE solution, and the reactions were performed at 35 C and pH 5.0. After completion of hydrolysis, individual catechins and GA were analyzed by HPLC, based on a comparison of authentic reference standards. The levels of EGCG and GA as reactant and products, respectively, were indicated on a graph (A), and the quantity of each catechins was estimated from the integrated data (B).
polyphenols, such as tannic acid, gallotannin, catechin, and proanthocyanidin, can react with proteins to form a stabilized complex, resulting in the formation of sediment and haze. This property of polyphenols results in the change of tea-based enzyme conformation, leading to a loss of catalytic activity. Many enzymes, such as tyrosinase, peroxidase, trypsin (22), decarboxylase (23), squalene epoxidase (24), and ribonuclease (25), were reported to be denatured by tea polyphenols. In our previous report, tea extract polyphenols also inhibited the activity of tannase, which catalyzes the bioconversion of catechins (5). We previously observed that the bioconversion of catechins by tannase was inhibited by elevated concentrations of substrates. Therefore, for bioconversion of catechins, tannases must be used under conditions in which substrateinduced inhibition is avoided. In this study, we investigated the effect of two types of tannases on bioconversion of catechins and substrate inhibition in the presence of inhibitors with the purpose of identifying an enzyme unrestricted from inhibition by tea extract or EGCG. The degallating activity of T2 tannase was not inhibited by high levels of tea extract and EGCG, whereas T1 tannase activity was inhibited by 2% tea extract and 10 mg mL1 EGCG (Figs. 1 and 2). Our data suggested that T2 tannase possesses an improved bioconversion ability of catechins with higher tolerance of substrate inhibitory concentrations than T1 tannase. Tolerance of substrate inhibition is one of the desired characteristics of enzymes for bioconversion reaction (26). Tannase activity has typically been
observed in bacteria or fungi, and tannase production has been achieved in several fermentation systems (14,27,28). The tolerance of tannase on tannins concentrations has been usually known to be observed in fungi Aspergillus species (29). Tannases used in this study were also derived from A. niger, but two tannases showed the difference on bioconversion activity due to different substrate inhibitory effect. This differential substrate inhibition could be happened on the same enzyme depending on various factors. Generally, fungi have been known to produce different forms of the same enzyme depending on the environmental conditions (30). Riou et al. (30) showed that two forms of bgalactosidases from Aspergillus oryzae were isolated with a big difference on glucose tolerance, and reported that such a difference was associated with carbon sources in the media. In addition, original habitat, where obtained these fungi, could provide an adaptational benefit, which is the capacity to grow in the high concentration of antimicrobial compounds like tannins. These environmental differences on strain growth could cause biochemical differences of same enzyme. One of the biochemical differences could be a hydrophobicity of the enzyme. Since interaction of tannin and protein (tannase) has been known to be based on the hydrophobic bonding (31,32), different hydrophobicities of two enzymes could allow the difference of bioconversion ability derived from different substrate inhibitions. Another biochemical difference of two enzymes could be a covalent modification like glycosylation, which can affect enzyme conformation and activity.
FIG. 7. Effect of T2 tannase treatment on DPPH (A) and ABTS (B) radical-scavenging activity of GTE. The half-maximal inhibitory concentration (IC50) value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50%.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
VOL. xx, 2014 Glycosylation has been known to affect the stability and solubility of the enzyme (33). Benoit et al. (34) showed that glycoside moiety stabilizes disulfide bonds of the proteins. In reality, A. niger tannases derived from different production methods were observed to have different thermal and chemical stability with a disparity of glycosylation (33). Thus, various biochemical properties including hydrophobicity, ion strength, and covalent modification would be analyzed in our next study. Since tannases used in this study were crude types, purity of the enzyme may be another concern, and materials (or carriers) mixed with enzymes could affect the enzymatic activity and stability. Carrier material is a major part of the immobilized enzymes, and its structure and property importantly affects enzymatic properties including tolerance to severe conditions (35,36). Gallic acid has been implicated in end-product inhibition of tannase (20). Adamczyk et al. (37) reported that pyrogallols, gallic acid, and gallaldehyde competitively inhibited the tannase of A. niger. Gallic acid was also an effective inhibitor of the tannase of Cryphonectria parasitica (38). Additionally, gallic acid was demonstrated to be an effective inhibitor of tannase when hamamelitannin was used as a substrate. Haslam et al. (39) indicated that gallic acid accumulation may inhibit tannase activity. Based on the above studies, gallic acid is suspected to regulate tannase activity by feedback inhibition. However, our data showed that degallating activity of T1 and T2 tannase was not affected by gallic acid addition (Fig. 3). These data suggest that degallating activity of T2 tannase was consistently exerted for the bioconversion of gallated catechins in the reaction (Fig. 3). However, if additional gallic acid content is applied to the reaction, tannase may be inhibited by gallic acid. Feedback inhibition of gallic acid will be examined with a broader range of concentrations in a subsequent study. One of the benefits of tannase-induced bioconversion may be an increase of functionality due to tannase treatment. In fact, green tea extract or its primary compound, EGCG, has been known to possess a variety of biological activities. Unfortunately, EGCG is the primary agent for the astringent taste green tea, so its unconsidered use may deteriorate the marketability of green tea. Transformation of EGCG by tannase activity can remove the tea’s astringency, albeit at the expense of EGCG’s biological benefit. Based on our data (Figs. 5 and 7), T2 tannase treatment displayed increased radical-scavenging activity compared with untreated sample. This result indicates that tannase T2 treatment would not remove the antioxidative effects of EGCG. The radical-scavenging effects of tannase hydrolysates appear to be due to degallated catechins, such as EGC, GC, and EC, which continue to possess radical-scavenging activity. Lu and Chen (16) reported that tannase-catalyzed hydrolysis of gallated catechins (such as EGCG and ECG) increased the radical-scavenging activity of tea extracts against superoxide anion, hydrogen peroxides, and DPPH. In addition, Battestin et al. (40) displayed a positive association between radical-scavenging capacity and an increase of EGC and gallic acid content in green tea extracts. However, Guo et al. (41) reported that the scavenging effects of gallated catechins (EGCG and GCG) on free radicals were stronger than those of degallated catechins (EGC, GC, EC, (þ)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (þ)-C. EGCG is the primary constituent of tea catechins, accounting for approximately 40% of the total polyphenol content in green tea (19). Although EGCG is the best-known chemoprevention agent among tea catechins, several concerns exist with respect to its potential capacity to cause DNA damage. For instance, EGCGinduced DNA damage can occur in the presence of metals complexes such as Cu2þ and Fe3þ. Specifically, in the presence of Fe3þeEDTA, DNA-damage severity occurs in the order of EGCG > EGC > ECG >> catechin (42). Tea, an edible or medicated beverage consumed since ancient times, remains the subject of attention due to its biological benefits,
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such as antioxidant activity. However, taste palatability of tea is recognized as an important criterion in tea consumption, and the detanninification of tanniniferous foods, beverages, feed, and fodder has been attempted on an industrial scale. Bioconversion of tea catechins via tannase treatment is one of such attempts to increase the palatability and quality of tea products without the concomitant loss of biological benefit. In conclusion, Tannase is an enzyme with a variety of potential biotechnological applications. However, many of these applications have been limited owing to the inhibitory effect of catechins. Our data suggest that T2 tannase is a useful biotransformation agent of tea catechins. In addition, T2 tannasecatalyzed hydrolysis of the catechin gallates (EGCG and ECG) in green tea increases the antioxidant activities of the resultant hydrolysates in the absence of any inhibition. Thus, our study suggests that T2 tannase treatment has a possible commercial application based on its role in the improved quality and functionality of tea products. References 1. Ramdani, D., Chaudhry, A. S., and Seal, C. J.: Chemical composition, plant secondary metabolites, and minerals of green and black teas and the effect of different tea-to-water ratios during their extraction on the composition of their spent leaves as potential additives for ruminants, J. Agric. Food Chem., 61, 4961e4967 (2013). 2. Chen, C., Shen, G., Hebbar, V., Hu, R., Owuor, E. D., and Kong, A. N.: Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells, Carcinogenesis, 24, 1369e1378 (2003). 3. Lekha, P. K. and Lonsane, B. K.: Production and application of tannin acyl hydrolase: state of the art, Adv. Appl. Microbiol., 44, 215e260 (1997). 4. Niho, N., Shibutani, M., Tamura, T., Toyoda, K., Uneyama, C., Takahashi, N., and Hirose, M.: Subchronic toxicity study of gallic acid by oral administration in F344 rats, Food Chem. Toxicol., 39, 1063e1070 (2001). 5. Noh, D. O., Choi, H.-S., and Suh, H. J.: Catechine biotransformation by tannase with sequential addition of substrate, Process Biochem., 49, 271e276 (2014). 6. Aguilar, C. N. and Gutiérrez-Sánchez, G.: Review: sources, properties, applications and potential uses of tannin acyl hydrolase, Food Sci. Technol. Int., 7, 373e382 (2001). 7. Aguilar, C. N., Rodriguez, R., Gutierrez-Sanchez, G., Augur, C., FavelaTorres, E., Prado-Barragan, L. A., Ramirez-Coronel, A., and ContrerasEsquivel, J. C.: Microbial tannases: advances and perspectives, Appl. Microbiol. Biotechnol., 76, 47e59 (2007). 8. Lu, M.-J. and Chen, C.: Enzymatic tannase treatment of green tea increases in vitro inhibitory activity against N-nitrosation of dimethylamine, Process Biochem., 42, 1285e1290 (2007). 9. Chávez-González, M., Rodríguez-Durán, L. V., Balagurusamy, N., PradoBarragán, A., Rodríguez, R., Contreras, J. C., and Aguilar, C. N.: Biotechnological advances and challenges of tannase: an overview, Food Bioprocess Technol., 5, 445e459 (2012). 10. Hong, Y. H., Jung, E. Y., Park, Y., Shin, K. S., Kim, T. Y., Yu, K. W., Chang, U. J., and Suh, H. J.: Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extracts, Biosci. Biotechnol. Biochem., 77, 22e29 (2013). 11. Albertse, E. H.: Cloning, expression and characterization of tannase from Aspergillus species. MS thesis. University of the Free State, South Africa (2002) . 12. Bi, S., He, X., and Haslam, E.: Gelatin-polyphenol interaction, J. Am. Leather Chem. Assoc., 89, 98e104 (1995). 13. Spencer, C. M., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P. N., Magnolato, D., Lilley, T. H., and Haslam, E.: Polyphenol complexationdsome thoughts and observations, Phytochemistry, 27, 2397e2409 (1988). 14. Aguilar, C. N., Augur, C., Favela-Torres, E., and Viniegra-Gonzalez, G.: Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid, J. Ind. Microbiol. Biotechnol., 26, 296e302 (2001). 15. Aguilar, C. N., Augur, C., Favela-Torres, E., and Viniegra-González, G.: Induction and repression patterns of fungal tannase in solid-state and submerged cultures, Process Biochem., 36, 565e570 (2001). 16. Lu, M.-J. and Chen, C.: Enzymatic modification by tannase increases the antioxidant activity of green tea, Food Res. Int., 41, 130e137 (2008). 17. Cheung, L., Cheung, P. C., and Ooi, V. E.: Antioxidant activity and total phenolics of edible mushroom extracts, Food Chem., 81, 249e255 (2003). 18. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and RiceEvans, C.: Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med., 26, 1231e1237 (1999). 19. Stoner, G. D. and Mukhtar, H.: Polyphenols as cancer chemopreventive agents, J. Cell. Biochem., 59, 169e180 (1995).
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20. Bradoo, S., Gupta, R., and Saxena, R.: Parametric optimization and biochemical regulation of extracellular tannase from Aspergillus japonicus, Process Biochem., 32, 135e139 (1997). 21. Wu, C. D. and Wei, G.-X.: Tea as a functional food for oral health, Nutrition, 18, 443e444 (2002). 22. Huang, H. H., Kwok, K. C., and Liang, H. H.: Effects of tea polyphenols on the activities of soybean trypsin inhibitors and trypsin, J. Sci. Food Agric., 84, 121e126 (2004). 23. Bertoldi, M., Gonsalvi, M., and Voltattorni, C. B.: Green tea polyphenols: novel irreversible inhibitors of dopa decarboxylase, Biochem. Biophys. Res. Commun., 284, 90e93 (2001). 24. Abe, I., Seki, T., Umehara, K., Miyase, T., Noguchi, H., Sakakibara, J., and Ono, T.: Green tea polyphenols: novel and potent inhibitors of squalene epoxidase, Biochem. Biophys. Res. Commun., 268, 767e771 (2000). 25. Ghosh, K. S., Maiti, T. K., and Dasgupta, S.: Green tea polyphenols as inhibitors of ribonuclease A, Biochem. Biophys. Res. Commun., 325, 807e811 (2004). 26. Raj, J., Seth, A., Prasad, S., and Bhalla, T. C.: Bioconversion of butyronitrile to butyramide using whole cells of Rhodococcus rhodochrous PA-34, Appl. Microbiol. Biotechnol., 74, 535e539 (2007). 27. Sharma, S., Agarwal, L., and Saxena, R. K.: Statistical optimization for tannase production from Aspergillus niger under submerged fermentation, Indian J. Microbiol., 47, 132e138 (2007). 28. Rodriguez, H., de las Rivas, B., Gomez-Cordoves, C., and Munoz, R.: Characterization of tannase activity in cell-free extracts of Lactobacillus plantarum CECT 748T, Int. J. Food Microbiol., 121, 92e98 (2008). 29. Cruz-Hernández, M., Augur, C., Rodríguez, R., Contreras-Esquivel, J. C., and Aguilar, C. N.: Evaluation of culture conditions for tannase production by Aspergillus niger GH1, Food Technol. Biotechnol., 44, 541e544 (2006). 30. Riou, C., Salmon, J. M., Vallier, M. J., Gunata, Z., and Barre, P.: Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae, Appl. Environ. Microbiol., 64, 3607e3614 (1998). 31. de Freitas, V., Carvalho, E., and Mateus, N.: Study of carbohydrate influence on proteinetannin aggregation by nephelometry, Food Chem., 81, 503e509 (2003).
J. BIOSCI. BIOENG., 32. Oh, H. I., Hoff, J. E., Armstrong, G. S., and Haff, L. A.: Hydrophobic interaction in tannin-protein complexes, J. Agric. Food Chem., 28, 394e398 (1980). 33. Renovato, J., Gutiérrez-Sánchez, G., Rodríguez-Durán, L. V., Bergman, C., Rodríguez, R., and Aguilar, C. N.: Differential properties of Aspergillus niger tannase produced under solid-state and submerged fermentations, Appl. Biochem. Biotechnol., 165, 382e395 (2011). 34. Benoit, I., Asther, M., Sulzenbacher, G., Record, E., Marmuse, L., Parsiegla, G., Gimbert, I., Asther, M., and Bignon, C.: Respective importance of protein folding and glycosylation in the thermal stability of recombinant feruloyl esterase A, FEBS Lett., 580, 5815e5821 (2006). 35. Tischer, W. and Wedekind, F.: Immobilized enzymes: methods and applications, pp. 95e126, in: de Meijere, A., Houk, K. N., Kessler, H., Lehn, J.-M., Ley, S. V., Schreiber, S. L., Thiem, J., Trost, B. M., Vogtle, F., and Yamamoto, H. (Eds.), Biocatalysis-from discovery to application. Springer, Berlin (1999). 36. Zhang, D.-H., Yuwen, L.-X., and Peng, L.-J.: Parameters affecting the performance of immobilized enzyme, J. Chem., 2013, 1e7 (2013). 37. Adamczyk, S., Kiikkila, O., Kitunen, V., and Smolander, A.: Potential response of soil processes to diterpenes, triterpenes and tannins: nitrification, growth of microorganisms and precipitation of proteins, Appl. Soil. Ecol., 67, 47e52 (2013). 38. Farias, G., Gorbea, C., Elkins, J., and Griffin, G.: Purification, characterization, and substrate relationships of the tannase from Cryphonectria parasitica, Physiol. Mol. Plant Pathol., 44, 51e63 (1994). 39. Haslam, E., Haworth, R., Jones, K., and Rogers, H.: Gallotannins. Part I. Introduction: and the fractionation of tannase, J. Chem. Soc. (Resumed), 1829e1835 (1961). 40. Battestin, V., Macedo, G., and De Freitas, V.: Hydrolysis of epigallocatechin gallate using a tannase from Paecilomyces variotii, Food Chem., 108, 228e233 (2008). 41. Guo, Q., Zhao, B. L., Shen, S. R., Hou, J. W., Hu, J. G., and Xin, W. J.: ESR study on the structure-antioxidant activity relationship of tea catechins and their epimers, Biochim. Biophys. Acta, 1427, 13e23 (1999). 42. Furukawa, A., Oikawa, S., Murata, M., Hiraku, Y., and Kawanishi, S.: (-)-Epigallocatechin gallate causes oxidative damage to isolated and cellular DNA, Biochem. Pharmacol., 66, 1769e1778 (2003).
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract Joo Hyun Baik,1, z Hyung Joo Suh,2, 3, z So Young Cho,2 Yooheon Park,2 and Hyeon-Son Choi2, * Cosmax Bio INC, Jecheon 390-250, Republic of Korea,1 Department of Food and Nutrition, Korea University, Seoul 136-703, Republic of Korea,2 and Department of Public Health Science, Graduate School, Korea University, Seoul 136-7033, Republic of Korea3 Received 11 February 2014; accepted 16 April 2014 Available online xxx
Tannases are important enzymes in the antioxidant potential of tea leaves. In this study, we evaluated the effect of two tannases (T1 and T2) on biotransformation of tea polyphenols and antioxidative activities from catechins in green tea extract (GTE). The T1 tannase-catalyzed reaction was inhibited by the addition of >2.0% GTE substrate, whereas the T2catalyzed reaction was not inhibited, even by addition of 5.0% GTE. Furthermore, the T1 tannase-catalyzed reaction was inhibited by addition of 10 mg mLL1 EGCG, whereas the T2 tannase-catalyzed reaction did not display any inhibitory effect. These results indicate that T2 tannase was more tolerant than T1 tannase to substrate inhibition in degallation reactions. Specifically, the substrate EGCG (90,687.1 mg mLL1) was transformed into gallic acid (50,242.9 mg mLL1) and EGC (92,598.3 mg mLL1) after 1-h treatment with T2 tannase (500 U gL1). The tannase-mediated product displayed higher in vitro radical-scavenging activity than the control. IC50 value of GTE on ABTS and DPPH radicals (46.1 mg mLL1 and 18.4 mg mLL1, respectively) decreased markedly after T2 tannase treatment (to 35.8 mg mLL1 and 15.1 mg mLL1, respectively). These results indicate that T2 tannase treatment of GTE enhanced its radical-scavenging activity, an increase that was also observed in the reaction using EGCG substrate. Taken together, our results revealed that T2 tannase is more suitable for biotransformation of catechins in GTE than T1 tannase, and T2 treatment provides an enhanced radical-scavenging effect. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Tannase; Biotransformation; Tea catechins; Antioxidative activity; Substrate inhibition]
Tea polyphenols constitute one of several essential components of tea that affect physiological functions. Catechins are the primary polyphenols found in tea, accounting for 75e80% of its soluble ingredients (1), including epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC), gallocatechin (GC) and catechin (C). EGCG constitutes over 50% of catechin content in tea (2). Tannase or tannin acyl hydrolase (EC, 3.1.1.20) catalyzes the hydrolysis of ester and depside bonds present in hydrolyzable tannins or gallic acid esters, such as EGCG or ECG, thereby releasing glucose or gallic acid as products (3). The release of gallic acid is physiologically beneficial due to its significant antioxidant potency (4). We previously reported that increase of gallic acid levels by tannase treatment was associated with an increase in antioxidant activity (5). Gallic acid is used in the synthesis of propyl gallate, which is an antioxidant primarily found in fats and oils, as well as in beverages. Tannase is also extensively used in the preparation of instant tea, acorn wine, and coffee-flavored soft drinks; the clarification of beer and fruit juices; and the detannification of foods (3,6,7). Many studies of tannase-mediated biotransformation of tea catechins have been performed (5,8,9). Our previous study (10)
* Corresponding author. Tel.: þ82 2 940 2764; fax: þ82 2 940 2859. E-mail address: [email protected] (H.-S. Choi). z The first two authors contributed equally to this work.
reported that tea catechins (such as EGCG or ECG) were hydrolyzed by tannase action (to generate EGC or EC, respectively, as well as gallic acid) by using >1% tea extract as a substrate. However, biotransformation of catechins by tannase was observed to be affected by substrate-mediated dose-dependent inhibition (5). Albertse (11) also reported the inhibition of tannase at higher concentrations of EGCG. Tea polyphenols, such as EGCG, are known to be involved in enzyme inhibition as well as the formation of precipitants in tea drinks (12,13). Substrate-mediated inhibition has been an important limitation on the widespread use of tannases. Therefore, the discovery of a tannase that is not susceptible to substrate inhibition is an important goal for the use of tannases in the biotransformation of tea catechins. At the present, many companies manufacture tannases of varying catalytic properties (7) using multiple methods. Tannases from Kikkoman (Japan) and Biocon (India) are manufactured by solid-state fermentation (SSF). The reactivity of tannase on substrates can depend on the enzyme source, culture methods, degree of purification, etc. (7). In general, tannases have been isolated from microbial sources, such as fungi, using SSF or submerged fermentation (SmF). Several differences in tannase production from microbes have been observed between SSF and SmF methods (14,15). For example, substrate concentrations are recognized to affect the regulatory mechanisms of tannase production (14,15). In this study, we examined the effect of two fungi-derived tannases from different manufacturers on biotransformation of tea
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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catechins and substrate inhibition. The antioxidative effects of tannase hydrolysates between the two types of tannases were also compared. This study shows that T2 tannase treatment leads to vastly improved biotransformation of tea catechins over T1 tannase treatment, without concomitant substrate inhibition compared. MATERIALS AND METHODS Materials and chemicals Green tea extract (GTE) was purchased from Zhejiang Huang Minghuang Natural Products Development Co., Ltd. (Beijing, China). The extract was determined by high-performance liquid chromatography (HPLC) to be composed of 71.9% catechins, including 40.9% EGCG. Acetic acid and acetonitrile (HPLC analytical grade) were purchased from Fisher Scientific (Pittsburgh, PA, USA). HPLC-grade standard gallic acid (GA), EGC, EC, EGCG, ECG, and standards were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). The remaining reagents used were of analytical grade. Tannases were purchased from Kikkoman Corporation (Tokyo, Japan) and Bision Biochem. Co. (Sungnam, Republic of Korea). Tannase treatment and determination of catechins by HPLC Thirty mg of tannase (500 U g1) was added to 50 mL of 1% GTE solution, and the reactions were performed at 35 C. The tannase reactant of green tea was obtained at regular intervals until the completion of hydrolysis (10). The inactivation of hydrolysis was performed by heating the sample at 100 C for 10 min after reaction. The content of each catechin was measured using an HPLC system equipped with a hypersil C18 column (5 mm, 25 0.46 cm ID) and a UVeVIS detector. The mobile phase contained 1% acetic acid (solvent A) and acetonitrile (solvent B) with a linear gradient starting at an A/B ratio of 92/8 and completing at 73/27 over a 40-min period at a flow rate of 1 mL min1. Identification of individual catechins and GA was based on a comparison of the retention times of the sample peaks with those of authentic reference standards. The quantity of each constituent in green tea was estimated from the integrated data (16). Inhibitory activity of EGCG and gallic acid The effect of EGCG and gallic acid addition was tested with GTE. Gallic acid (5 and 10 mg mL1) and EGCG (1, 2, 5 and 10 mg mL1) were added to the enzymatic reaction mixture (50 mL) of tannase. Thirty mg of tannase (500 U g1) was added to 50 mL of 1% GTE solution, and the reactions were performed at 35 C for 2 h. The inactivation of hydrolysis was performed by heating at 100 C for 10 min after reaction. Samples were removed at regular intervals until the completion of hydrolysis and assayed to determine changes in the level of catechins.
Scaleup for the biotransformation of GTE Six gram of tannase was added to 10 L of 10% GTE solution, with the reactions performed at 35 C. The tannase reactant of green tea was obtained at regular intervals until the completion of hydrolysis. The inactivation of hydrolysis was performed by heating at 100 C for 10 min after the reaction. The reactant was lyophilized and used for the assay of catechin and radical-scavenging activity. Antioxidant activities of green tea extract The scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured, according to the method of Cheung et al. (17) The scavenging activity of the 2,2-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical was measured according to the method of Re et al. (18) The DPPH and ABTS radical-scavenging activities were calculated as follows: Radical scavenging activityð%Þ ¼
. 1 Asample Acontrol 100
(1)
where Asample is the absorbance in the presence of sample, and Acontrol is the absorbance in the absence of sample. The half-maximal inhibitory concentration (IC50) value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50%. Statistical analysis All expressed values are mean values of triplicate determinations. All statistical analyses were performed using the Statistical Package for Social Sciences version 12.0 (SPSS, Chicago, IL, USA). Differences among the samples were evaluated statistically by one-way analysis of variance (ANOVA) and Tukey’s New Multiple Test. All data were two-sided at a 5% significance level, and have been reported as mean deviation (SD).
RESULTS Effect of GTE substrate concentration on tannase T1 and T2 are Aspergillus niger-derived tannases produced using different methods. T1 tannase obtained from Kikkoman was produced by SSF, while T2 tannase from Bision was produced by SmF. To measure the effect of substrate concentration in tannase T1- and T2mediated reactions, several concentrations of green tea extract (0.5e5.0%) were added to the reaction mixture, followed by HPLC analysis of their products (Fig. 1). The addition of 0.5% GTE resulted in an increase of the quantity of gallic acid as a product of the T1
FIG. 1. Effect of GTE concentration on tannase T1 (A, B) and T2 (C, D) activity. The reactions were performed at 35 C and pH 5.0 at various GTE concentrations.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
VOL. xx, 2014 and T2 tannase-mediated reaction after a 1-h incubation period. The level of gallic acid generated from the T2 tannase-mediated reaction was slightly higher than that of the T1 tannase-mediated reaction with 0.5% GTE; T1 tannase generated 1600 mg mL1 gallic acid, while T2 tannase generated 1800 mg mL1 gallic acids after a 1-hr incubation period (Fig. 1). Conversely, the quantity of EGCG, a substrate of the T1 and T2 tannase reaction, decreased to negligible levels after the 1-h reaction period, indicating that almost all of the EGCG was utilized for enzyme-catalyzed conversion into gallic acid. When 1.0% GTE was added, the T1 enzyme reaction proceeded at a slower rate than the T2 enzyme reaction. In the T2 reaction, gallic acid production was achieved up to a gallic acid concentration of 3000 mg mL1 within 1 h, whereas the T1 tannase reaction required 3 h to generate gallic acid to a concentration of 3000 mg mL1. Furthermore, gallic acid production by the T1 reaction was greatly decreased in the treatment of >2.0% GTE, suggesting that the T1 tannase reaction was subject to inhibition at these substrate levels. T2 tannase generated gallic acid concentrations of 6000 mg mL1 in a 1-h period, consuming all quantities of EGCG within 30 min, suggesting that T2 tannase was not subject to substrate inhibition at 2% GTE, displaying a higher enzymatic activity in the same substrate than T1. Even the addition of 5% GTE did not inhibit T2 tannase-catalyzed gallic acid production, whereby all quantities of EGCG in GTE were consumed within 2 h. Thus, T2 tannase led to a larger quantity of product in a shorter reaction time compared with T1 tannase. Our data indicated that SmF-derived T2 tannase has a much greater capacity for conversion of EGCG into gallic acid than SSF-derived T1 tannase. Effect of EGCG concentration as substrate on tannase Fresh green tea leaves typically contain 36% polyphenols, primarily catechins. EGCG is the predominant catechin
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present in green tea leaves (48e55% of total polyphenols) (19). The substrate GTE is composed of 71.9% catechins, including 40.9% EGCG, which may be responsible for the inhibitory effect of GTE in the tannase reaction. Therefore, we examined the effect of EGCG concentrations as a tannase inhibitor on both tannase T1 and T2 reactions (Fig. 2). The addition of 1 and 2 mg mL1 EGCG did not inhibit T1 and T2 tannase reactions in which gallic acid product levels were measured at 500 mg mL1 and 1000 mg mL1, respectively. Additionally, 1 and 2 mg mL1 of added EGCGs were completely consumed by 10 min in both tannase reactions. This result demonstrated that most EGCG was consumed for conversion into gallic acid within the 30-min reaction period catalyzed by T1 and T2 tannase (Fig. 2). Addition of 5 mg mL1 EGCG did not inhibit T1 or T2 tannases, but displayed the difference in rate of product generation between the two tannases. T1 tannase activity generated 2000 mg mL1 of gallic acid from 5 mg mL1 of EGCG within a 1-h period, while T2 tannase activity generated 2400 mg mL1 gallic acid within 10 min. In addition, EGCG levels in the T1 tannase reaction were decreased in a 2-h period, whereas the T2 tannase reaction induced a dramatic reduction of EGCG levels within 10 min. These results demonstrated that the enzymatic activity of T2 tannase was much greater than that of T1, consistent with the above GTE data (Fig. 1). Specifically, T1 tannase was completely inhibited by the addition of 10 mg mL1 EGCG without any concomitant consumption of substrate, whereas T2 tannase consumed 10 mg mL1 EGCG within 10 min to generate high levels (5000 mg mL1) of gallic acids. Our data indicated that T2 tannase apparently has stronger enzymatic capabilities than T1 tannase to convert EGCG into gallic acid, and is much more tolerant to EGCG inhibition.
FIG. 2. Effect of EGCG concentration on tannase T1 (A, B) and T2 (C, D) activity. The reactions were performed at 35 C and pH 5.0 at various EGCG concentrations in 50 mL of total volume.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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Effect of gallic acid concentration as product on tannase Gallic acid is reported to be associated with end-product repression of tannase (20). We subsequently examined the inhibitory effect of gallic acid addition on T1 and T2 tannases. When 5 and 10 mg mL1 of gallic acid was added to the reactions, gallic acid product levels increased, while EGCG levels decreased, in a 2-h reaction period compared with 0 time (control; Fig. 3). Reactions containing 5 and 10 mg mL1 gallic acid additions increased gallic acid levels by 120% and 73%, respectively. The increase width of gallic acid product in these reactions was lower than untreated control, due to the higher initial levels of gallic acid present after addition. However, three reaction groups (0, 5, and 10 mg mL1 of gallic acid) displayed similar levels of consumed EGCG during the reactions (Fig. 3), a trend that was observed in both tannases. These results demonstrated that the tannases T1 and T2 were not inhibited by the addition of gallic acids (5 and 10 mg mL1). Our data suggested that addition of gallic acid did not induce the endproduct repression of T1 and T2 tannases. Moreover, the addition of gallic acid also reduced the pH of the sample from 6.0 to 3.8 (data was not shown), although the decease of pH did not influence the T1 and T2 reaction. EGCG biotransformation by T2 tannase and radicalscavenging activity As the most abundant polyphenol in green tea, EGCG is one of the most interesting components of tea leaf, exhibiting potent antioxidant activity in vitro and in vivo (21). Additionally, tea polyphenols such as catechins (including EGCG) and gallic acid have also been considered primary effectors in the beneficial effects of tea on the human health. However, EGCG has been reported to present potential risks, including DNA damage, and is a primary ingredient providing an astringent taste to tea drink.
J. BIOSCI. BIOENG., Therefore, we transformed EGCG into degallated catechins with the catalytic action of T2 tannase and examined the antioxidative effects of resulting EGCG hydrolysates. EGCG and its hydrolysates, gallic acid and EGC, were analyzed with HPLC (Fig. 4). EGCG (90,687.1 mg mL1) was completely transformed into EGC (92,598.3 mg mL1) and gallic acid (50,242.9 mg mL1) after T2 tannase treatment for 1 h, as observed in the post-reaction HPLC chromatogram (Fig. 4). Next, we examined the free-radical scavenging activities between before and after T2 tannase reaction using the DPPH and ABTS method. Radical-scavenging capacity was proportionally increased with EGC and gallic acid contents in the bioconversion reaction. Scavenging capabilities on DPPH and ABTS radicals were also significantly enhanced after T2 tannase treatment (Fig. 5); IC50 values on DPPH and ABTS radicals after T2 treatment (14.0 and 25.5 mg mL1, respectively) were significantly lower than those before T2 treatment (17.8 and 39.6 mg mL1, respectively; P < 0.05). This result demonstrated that T2 tannase treatment increased radical-scavenging activity of the EGCG solution via enzymatic biotransformation. Increased levels of EGC and gallic acids were suspected to be responsible for the enhancement of radical-scavenging activity. Our data indicated that tannase-derived bioconversion could improve the biological function of green tea extracts. Scale-up for the biotransformation of GTE and radicalscavenging activity of biotransformed GTE We examined the availability of T2 tannase for industrial bioconversion of GTE. Total reaction volume was scaled up to 10 L, with increased T2 tannase and substrate GTE. Ten percent GTE solution was treated with T2 tannase for a 4-h period. T2 tannase treatment resulted in an increase in the amount of gallic acid product of the tannase reaction from 2480.0 mg mL1 to 39,797.5 mg mL1 after a 2-h reaction period (Fig. 6A). Moreover, the amount of EGCG substrate of the tannase
FIG. 3. Effect of gallic acid concentration on tannase T1 (A, B) and T2 (C, D) activity. The reactions were performed at 35 C and pH 5.0 at various gallic acid concentrations.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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FIG. 4. EGCG (A) biotransformation to EGC and gallic acid (B) by tannase T2. The reactions (50 mL) were performed at 35 C and pH 5.0 with EGCG (10 mg) as a substrate and tannase T2 (30 mg). Catechin was measured using an HPLC system equipped with a hypersil C18 column (5 mm, 25 0.46 cm ID) and a UVeVIS detector. The mobile phase consisted of 1% acetic acid (solvent A) and acetonitrile (solvent B) with a linear gradient starting at an A/B ratio of 92/8 and terminating at 73/27 over a 40-min period at a flow rate of 1 mL min1.
reaction decreased from 39,661.5 mg mL1 to 6.5 mg mL1 after a 2-h reaction period. Gallic acid production sharply increased with T2 treatment until 1 h and then slightly increased thereafter. The increasing trend of gallic acid was precisely associated with the downward trend of EGCG (Fig. 6A). After 2 h of T2 tannase treatment, the sample displayed a decrease in gallated catechin levels, including EGCG, ECG, and GCG, with a concomitant increase in degallated catechin levels including EGC, EC, and GC. This result demonstrated that most EGCG, ECG, and GCG were converted to EGC, EC, and GC, respectively, after a 2h reaction period (Fig. 6B). At 2 h, EGC, EC, and GC content increased from 15,149.2, 5696.7, and 2065.4 mg mL1 to 53,341.8, 14,576.9, and 3638.8 mg mL1, respectively. Our data indicated that degallation of T2 tannase functioned normally for biotransformation of catechins in a scaled-up system. The radical-scavenging activity of GTE was determined before and after T2 tannase treatment in a scaled-up system. As shown in
Fig. 7, the IC50 value of GTE for DPPH radical-scavenging was decreased from 18.4 mg mL1 to 15.1 mg mL1 by T2 tannase treatment. In addition, the IC50 of GTE on ABTS radicals was decreased to 35.8 g mL1 from 46.1 g mL1 by T2 tannase treatment. These data demonstrated that T2 tannase treatment improved the antioxidative effects of GTE via biotransformation of catechins, consistent with above data reporting a higher antioxidative activity with T2 tannase treatment in EGCG solution (Fig. 5). Our results suggested that T2 tannase may play an important role in the conversion of catechins in an industrial setting.
DISCUSSION Tea polyphenols play an important role in protein precipitation and enzyme inhibition through the formation of a variety of complexes with tea ingredients (12,13). It is known that most
FIG. 5. Effect of T2 tannase treatment on DPPH (A) and ABTS (B) radical-scavenging activity of EGCG. The half-maximal inhibitory concentration (IC50) value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50%.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
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FIG. 6. GTE biotransformation by T2 tannase in scale-up system. Six gram of tannase (500 U g1) was added to 10 L of 10% GTE solution, and the reactions were performed at 35 C and pH 5.0. After completion of hydrolysis, individual catechins and GA were analyzed by HPLC, based on a comparison of authentic reference standards. The levels of EGCG and GA as reactant and products, respectively, were indicated on a graph (A), and the quantity of each catechins was estimated from the integrated data (B).
polyphenols, such as tannic acid, gallotannin, catechin, and proanthocyanidin, can react with proteins to form a stabilized complex, resulting in the formation of sediment and haze. This property of polyphenols results in the change of tea-based enzyme conformation, leading to a loss of catalytic activity. Many enzymes, such as tyrosinase, peroxidase, trypsin (22), decarboxylase (23), squalene epoxidase (24), and ribonuclease (25), were reported to be denatured by tea polyphenols. In our previous report, tea extract polyphenols also inhibited the activity of tannase, which catalyzes the bioconversion of catechins (5). We previously observed that the bioconversion of catechins by tannase was inhibited by elevated concentrations of substrates. Therefore, for bioconversion of catechins, tannases must be used under conditions in which substrateinduced inhibition is avoided. In this study, we investigated the effect of two types of tannases on bioconversion of catechins and substrate inhibition in the presence of inhibitors with the purpose of identifying an enzyme unrestricted from inhibition by tea extract or EGCG. The degallating activity of T2 tannase was not inhibited by high levels of tea extract and EGCG, whereas T1 tannase activity was inhibited by 2% tea extract and 10 mg mL1 EGCG (Figs. 1 and 2). Our data suggested that T2 tannase possesses an improved bioconversion ability of catechins with higher tolerance of substrate inhibitory concentrations than T1 tannase. Tolerance of substrate inhibition is one of the desired characteristics of enzymes for bioconversion reaction (26). Tannase activity has typically been
observed in bacteria or fungi, and tannase production has been achieved in several fermentation systems (14,27,28). The tolerance of tannase on tannins concentrations has been usually known to be observed in fungi Aspergillus species (29). Tannases used in this study were also derived from A. niger, but two tannases showed the difference on bioconversion activity due to different substrate inhibitory effect. This differential substrate inhibition could be happened on the same enzyme depending on various factors. Generally, fungi have been known to produce different forms of the same enzyme depending on the environmental conditions (30). Riou et al. (30) showed that two forms of bgalactosidases from Aspergillus oryzae were isolated with a big difference on glucose tolerance, and reported that such a difference was associated with carbon sources in the media. In addition, original habitat, where obtained these fungi, could provide an adaptational benefit, which is the capacity to grow in the high concentration of antimicrobial compounds like tannins. These environmental differences on strain growth could cause biochemical differences of same enzyme. One of the biochemical differences could be a hydrophobicity of the enzyme. Since interaction of tannin and protein (tannase) has been known to be based on the hydrophobic bonding (31,32), different hydrophobicities of two enzymes could allow the difference of bioconversion ability derived from different substrate inhibitions. Another biochemical difference of two enzymes could be a covalent modification like glycosylation, which can affect enzyme conformation and activity.
FIG. 7. Effect of T2 tannase treatment on DPPH (A) and ABTS (B) radical-scavenging activity of GTE. The half-maximal inhibitory concentration (IC50) value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50%.
Please cite this article in press as: Baik, J. H., et al., Differential activities of fungi-derived tannases on biotransformation and substrate inhibition in green tea extract, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.012
VOL. xx, 2014 Glycosylation has been known to affect the stability and solubility of the enzyme (33). Benoit et al. (34) showed that glycoside moiety stabilizes disulfide bonds of the proteins. In reality, A. niger tannases derived from different production methods were observed to have different thermal and chemical stability with a disparity of glycosylation (33). Thus, various biochemical properties including hydrophobicity, ion strength, and covalent modification would be analyzed in our next study. Since tannases used in this study were crude types, purity of the enzyme may be another concern, and materials (or carriers) mixed with enzymes could affect the enzymatic activity and stability. Carrier material is a major part of the immobilized enzymes, and its structure and property importantly affects enzymatic properties including tolerance to severe conditions (35,36). Gallic acid has been implicated in end-product inhibition of tannase (20). Adamczyk et al. (37) reported that pyrogallols, gallic acid, and gallaldehyde competitively inhibited the tannase of A. niger. Gallic acid was also an effective inhibitor of the tannase of Cryphonectria parasitica (38). Additionally, gallic acid was demonstrated to be an effective inhibitor of tannase when hamamelitannin was used as a substrate. Haslam et al. (39) indicated that gallic acid accumulation may inhibit tannase activity. Based on the above studies, gallic acid is suspected to regulate tannase activity by feedback inhibition. However, our data showed that degallating activity of T1 and T2 tannase was not affected by gallic acid addition (Fig. 3). These data suggest that degallating activity of T2 tannase was consistently exerted for the bioconversion of gallated catechins in the reaction (Fig. 3). However, if additional gallic acid content is applied to the reaction, tannase may be inhibited by gallic acid. Feedback inhibition of gallic acid will be examined with a broader range of concentrations in a subsequent study. One of the benefits of tannase-induced bioconversion may be an increase of functionality due to tannase treatment. In fact, green tea extract or its primary compound, EGCG, has been known to possess a variety of biological activities. Unfortunately, EGCG is the primary agent for the astringent taste green tea, so its unconsidered use may deteriorate the marketability of green tea. Transformation of EGCG by tannase activity can remove the tea’s astringency, albeit at the expense of EGCG’s biological benefit. Based on our data (Figs. 5 and 7), T2 tannase treatment displayed increased radical-scavenging activity compared with untreated sample. This result indicates that tannase T2 treatment would not remove the antioxidative effects of EGCG. The radical-scavenging effects of tannase hydrolysates appear to be due to degallated catechins, such as EGC, GC, and EC, which continue to possess radical-scavenging activity. Lu and Chen (16) reported that tannase-catalyzed hydrolysis of gallated catechins (such as EGCG and ECG) increased the radical-scavenging activity of tea extracts against superoxide anion, hydrogen peroxides, and DPPH. In addition, Battestin et al. (40) displayed a positive association between radical-scavenging capacity and an increase of EGC and gallic acid content in green tea extracts. However, Guo et al. (41) reported that the scavenging effects of gallated catechins (EGCG and GCG) on free radicals were stronger than those of degallated catechins (EGC, GC, EC, (þ)-C), and the scavenging effects of EGC and GC were stronger than those of EC and (þ)-C. EGCG is the primary constituent of tea catechins, accounting for approximately 40% of the total polyphenol content in green tea (19). Although EGCG is the best-known chemoprevention agent among tea catechins, several concerns exist with respect to its potential capacity to cause DNA damage. For instance, EGCGinduced DNA damage can occur in the presence of metals complexes such as Cu2þ and Fe3þ. Specifically, in the presence of Fe3þeEDTA, DNA-damage severity occurs in the order of EGCG > EGC > ECG >> catechin (42). Tea, an edible or medicated beverage consumed since ancient times, remains the subject of attention due to its biological benefits,
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such as antioxidant activity. However, taste palatability of tea is recognized as an important criterion in tea consumption, and the detanninification of tanniniferous foods, beverages, feed, and fodder has been attempted on an industrial scale. Bioconversion of tea catechins via tannase treatment is one of such attempts to increase the palatability and quality of tea products without the concomitant loss of biological benefit. In conclusion, Tannase is an enzyme with a variety of potential biotechnological applications. However, many of these applications have been limited owing to the inhibitory effect of catechins. Our data suggest that T2 tannase is a useful biotransformation agent of tea catechins. In addition, T2 tannasecatalyzed hydrolysis of the catechin gallates (EGCG and ECG) in green tea increases the antioxidant activities of the resultant hydrolysates in the absence of any inhibition. Thus, our study suggests that T2 tannase treatment has a possible commercial application based on its role in the improved quality and functionality of tea products. References 1. Ramdani, D., Chaudhry, A. S., and Seal, C. J.: Chemical composition, plant secondary metabolites, and minerals of green and black teas and the effect of different tea-to-water ratios during their extraction on the composition of their spent leaves as potential additives for ruminants, J. Agric. Food Chem., 61, 4961e4967 (2013). 2. Chen, C., Shen, G., Hebbar, V., Hu, R., Owuor, E. D., and Kong, A. N.: Epigallocatechin-3-gallate-induced stress signals in HT-29 human colon adenocarcinoma cells, Carcinogenesis, 24, 1369e1378 (2003). 3. Lekha, P. K. and Lonsane, B. K.: Production and application of tannin acyl hydrolase: state of the art, Adv. Appl. Microbiol., 44, 215e260 (1997). 4. Niho, N., Shibutani, M., Tamura, T., Toyoda, K., Uneyama, C., Takahashi, N., and Hirose, M.: Subchronic toxicity study of gallic acid by oral administration in F344 rats, Food Chem. Toxicol., 39, 1063e1070 (2001). 5. Noh, D. O., Choi, H.-S., and Suh, H. J.: Catechine biotransformation by tannase with sequential addition of substrate, Process Biochem., 49, 271e276 (2014). 6. Aguilar, C. N. and Gutiérrez-Sánchez, G.: Review: sources, properties, applications and potential uses of tannin acyl hydrolase, Food Sci. Technol. Int., 7, 373e382 (2001). 7. Aguilar, C. N., Rodriguez, R., Gutierrez-Sanchez, G., Augur, C., FavelaTorres, E., Prado-Barragan, L. A., Ramirez-Coronel, A., and ContrerasEsquivel, J. C.: Microbial tannases: advances and perspectives, Appl. Microbiol. Biotechnol., 76, 47e59 (2007). 8. Lu, M.-J. and Chen, C.: Enzymatic tannase treatment of green tea increases in vitro inhibitory activity against N-nitrosation of dimethylamine, Process Biochem., 42, 1285e1290 (2007). 9. Chávez-González, M., Rodríguez-Durán, L. V., Balagurusamy, N., PradoBarragán, A., Rodríguez, R., Contreras, J. C., and Aguilar, C. N.: Biotechnological advances and challenges of tannase: an overview, Food Bioprocess Technol., 5, 445e459 (2012). 10. Hong, Y. H., Jung, E. Y., Park, Y., Shin, K. S., Kim, T. Y., Yu, K. W., Chang, U. J., and Suh, H. J.: Enzymatic improvement in the polyphenol extractability and antioxidant activity of green tea extracts, Biosci. Biotechnol. Biochem., 77, 22e29 (2013). 11. Albertse, E. H.: Cloning, expression and characterization of tannase from Aspergillus species. MS thesis. University of the Free State, South Africa (2002) . 12. Bi, S., He, X., and Haslam, E.: Gelatin-polyphenol interaction, J. Am. Leather Chem. Assoc., 89, 98e104 (1995). 13. Spencer, C. M., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P. N., Magnolato, D., Lilley, T. H., and Haslam, E.: Polyphenol complexationdsome thoughts and observations, Phytochemistry, 27, 2397e2409 (1988). 14. Aguilar, C. N., Augur, C., Favela-Torres, E., and Viniegra-Gonzalez, G.: Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid, J. Ind. Microbiol. Biotechnol., 26, 296e302 (2001). 15. Aguilar, C. N., Augur, C., Favela-Torres, E., and Viniegra-González, G.: Induction and repression patterns of fungal tannase in solid-state and submerged cultures, Process Biochem., 36, 565e570 (2001). 16. Lu, M.-J. and Chen, C.: Enzymatic modification by tannase increases the antioxidant activity of green tea, Food Res. Int., 41, 130e137 (2008). 17. Cheung, L., Cheung, P. C., and Ooi, V. E.: Antioxidant activity and total phenolics of edible mushroom extracts, Food Chem., 81, 249e255 (2003). 18. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and RiceEvans, C.: Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med., 26, 1231e1237 (1999). 19. Stoner, G. D. and Mukhtar, H.: Polyphenols as cancer chemopreventive agents, J. Cell. Biochem., 59, 169e180 (1995).
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