Elimination of hydrogen peroxide enhances tyrosinase-catalyzed synthesis of theaflavins

Process Biochemistry 85 (2019) 19–28

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Elimination of hydrogen peroxide enhances tyrosinase-catalyzed synthesis of theaflavins

T

Asako Narai-Kanayamaa, , Yuuka Uchidaa, Aya Kawashimaa, Tsutomu Nakayamaa,b ⁎

a b

Graduate School of Veterinary Medicine and Life Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

ARTICLE INFO

ABSTRACT

Keywords: Theaflavin Tyrosinase Enzymatic synthesis Hydrogen peroxide Catalase

In this study, we found that hydrogen peroxide was generated during the separate incubation of four catechins, (-)-epicatechin, (-)-epigallocatechin, and their galloylated forms, in the absence or presence of mushroom tyrosinase (EC 1.14.18.1). In particular, autooxidation of (-)-epigallocatechin and enzymatic oxidation of (-)-epicatechin gallate contributed to the increase of hydrogen peroxide. We confirmed the hydrogen peroxide-induced inactivation of tyrosinase and found that hydrogen peroxide was also generated during the tyrosinase-catalyzed synthesis of theaflavins by selectively combining two types of catechins, diphenol- and pyrogallol-types. Elimination of hydrogen peroxide by co-incubation with bovine catalase (EC 1.11.1.6) increased the products in synthetic reactions for TF1, TF2A, TF2B, and TF3 by 15%, 16%, 45%, and 18%, respectively. Based on the prospective mechanism of the tyrosinase-catalyzed synthesis of theaflavins, the yield of TF2B increased by elevating the initial ratio of (-)-epigallocatechin/(-)-epicatechin gallate in the reaction media. Furthermore, under such conditions, there were clear ameliorative effects of catalase on the tyrosinase-catalyzed synthesis of TF2B, reaching 3.1-fold increase of the product compared to the reaction without catalase. These results support a novel strategy to use both tyrosinase and catalase for the efficient synthesis of theaflavins from catechins.

1. Introduction Theaflavins, the characteristic orange or orange-red pigments in black tea leaves and their exudates, have recently attracted attention due to their various bioactivities, such as antioxidant activity against LDL oxidation [1], radical-scavenging activity [2], anticancer activity [3,4], antidiabetic effects [5], and inhibitory activity against bone loss in models of osteoporosis [6]. Theaflavins are produced during the fermentation of Camellia sinensis leaves, in which endogenous polyphenol oxidase (PPO) and peroxidase are involved [7,8]. Condensation of different pairs of catechins, one with a dihydroxylated B-ring (catechol-type) and the other with a trihydroxylated B-ring (pyrogalloltype), and a subsequent decarboxylation afford the formation of four kinds of theaflavins, theaflavin (TF1), theaflavin 3-O-gallate (TF2A), theaflavin 3′-O-gallate (TF2B), and theaflavin 3,3′-di-O-gallate (TF3) (Fig. 1) [9,10]. However, the concentration of theaflavins in black tea is fairly low, about 8–20 mg/100 mL of tea brewed from a teabag [11], due to their conversion into thearubigins by further oxidative polymerization during fermentation in black tea manufacture [12,13].

Therefore, synthetic reaction systems to produce sufficient amounts of theaflavins are necessary for further studies on their biological activities and applications. Some biosynthetic methods for the mass production of theaflavins have been reported using plant PPOs [9,13] or PPO purified from tea leaves [7,14,15] and C. sinensis cell culture [16]. We also reported the use of tyrosinase (EC 1.14.18.1) from the mushroom Agaricus bisporus, a metalloenzyme carrying two copper atoms at its active site with both monophenol monooxygenase and diphenol oxidase activities, as a commercially available PPO model [10]. During melanogenesis, tyrosinase controls two rate-limiting steps: the transformation of L-tyrosine to L-dopa (as a monophenol monooxygenase), and the conversion of dopa to dopaquinone (as a diphenol oxidase). Our previous study using a commercially available, purified tyrosinase showed that, in the enzymatic reactions with selectively combined catechins in aqueous buffer solution, TF1 can be more effectively synthesized than the other three theaflavins; however, yields of galloylated theaflavins, in particular TF2B and TF3, are very low [10]. Through kinetic analyses of tyrosinase-catalyzed reaction with (-)-epicatechin gallate (ECg) alone, a time-dependent decrease in the reaction rate was

Abbreviations: EC, (-)-epicatechin; EGC, (-)-epigallocatechin; ECg, (-)-epicatechin gallate; EGCg, (-)-epigallocatechin gallate; FOX, ferrous ion oxidation; H2O2, hydrogen peroxide; PPO, polyphenol oxidase; TF1, theaflavin; TF2A, theaflavin 3-O-gallate; TF2B, theaflavin 3′-O-gallate; TF3, theaflavin 3,3′-di-O-gallate ⁎ Corresponding author. E-mail address: [email protected] (A. Narai-Kanayama). https://doi.org/10.1016/j.procbio.2019.07.004 Received 21 January 2019; Received in revised form 13 June 2019; Accepted 2 July 2019 Available online 02 July 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Prospective mechanism of tyrosinase-catalyzed synthesis of theaflavins from catechins in aqueous conditions.

Fig. 2. Contents of H2O2 (A) and catechins (B) after incubation of respective catechins in 50 mM sodium phosphate buffer (pH 6.0) containing 10% ethanol in the absence (-) or presence (+) of tyrosinase at 0.1 mg/mL at 25 °C for 30 min. Data are means ± SD (n = 4). *Significantly different from the value at 0 min (P < 0.05).

observed despite the higher catalytic efficiency (kcat/KM) for ECg [10]. This implies suicide inactivation of the enzyme. There are several reports on suicide inactivation of tyrosinase, i.e., the enzyme is inactivated when it reacts with a substrate [17,18]. The proposed mechanism during its interaction with o-diphenol involves oxidation/reduction taking place on one of the two copper atoms in its active site, releasing o-quinone, copper (0), H2O2, and an inactive

enzyme [17,18]. In that case, the orientation of the phenyl ring in the enzyme-substrate complex is assumed to be approximately orthogonal to the plane defined by the copper and oxygen atoms. Therefore, the chemical structure of the substrates is important. Indeed, the kinetic constants that characterize the suicide inactivation of tyrosinase vary according to the phenolic substrate [17,18]. In addition, tyrosinase also has catalase activity, and its substrate 20

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Fig. 3. Time- and dose-dependent inactivation of tyrosinase by H2O2. After tyrosinase was pretreated with H2O2 at indicated concentrations, the enzyme was reacted with 4-methyl catechol with or without catalase and the oxidation activity was determined by a colorimetric assay. Values are relative to the activity of control tyrosinase which was untreated with H2O2. Data are means ± SD (n = 3).

Fig. 4. Effects of preincubation of tyrosinase with H2O2 on the enzyme activity for respective four catechins. After 0.2 mg/mL tyrosinase was pretreated without (-) or with (+) 100 μM H2O2 in 50 mM sodium phosphate buffer (pH 6.0) at 25 °C for 40 min, the enzyme solution was mixed with an equal volume of the same buffer containing 10 mM of each catechin and 0.2 mg/mL catalase. Data are means ± SD (n = 3).

H2O2 causes suicide inactivation, which is remarkable under anaerobic conditions but can still be noticed under aerobic ones [19]. In tyrosinase inactivation by H2O2, it is assumed that H2O2-induced oxidation of the two coppers in a deoxy form of the enzyme is not successfully concerted, forming an inactive enzyme that can no longer convert into the met- and oxy forms, which are the active enzyme species [19]. However, to our knowledge, there are no studies unequivocally describing the mechanism of H2O2-induced suicide inactivation of tyrosinase. It has been reported that H2O2 is produced during autooxidation of some phenolics including catechins [20–22] as well as by enzymatic oxidation of (+)-catechin [23]. In both cases, the addition of superoxide dismutase partially inhibits the generation of H2O2. Nakayama et al. measured H2O2 formed in aqueous solutions of tea catechins and proposed that gallocatechins such as (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCg) are oxidized by superoxide accompanied by the formation of H2O2 [20]. Through kinetic studies on the autooxidation of phenolic compounds and the effects of pH, Roginsky and Alegria [22] suggested that autooxidation is initiated by the reaction of molecular oxygen with a semiquinone-type radical derived from the substrate, and the resulting superoxide anion subsequently reacts with the substrate to generate a semiquinone radical and H2O2. Thus, non-enzymatic H2O2 generation from phenolics is dependent on their electron-donating abilities and the susceptibilities of semiquinone radicals to oxygen.

In the present study, we found the H2O2 generation during the tyrosinase-catalyzed reaction with (-)-epicatechin (EC), EGC, ECg or EGCg, the patterns of which were different among these four catechins, i.e., the H2O2 content in media significantly increased through the autooxidation of EGC and enzymatic oxidation of ECg. The resulting H2O2 inactivated tyrosinase depending on the incubation time and concentration. Furthermore, we examined the effects of H2O2 elimination by the addition of catalase (EC 1.11.1.6) from bovine liver on the tyrosinase-catalyzed synthesis of theaflavins from selectively combined catechins. As a result, the reaction rates and product yields in the presence of catalase were much higher compared to those of control reactions, in particular three times more TF2B was synthesized. 2. Materials and methods 2.1. Materials Four catechins, (-)-epicatechin (EC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCg), were kindly provided by Mitsui Norin (Shizuoka, Japan). Tyrosinase from A. bisporus (3130 units/mg solid) and catalase from bovine liver (11,000 units/mg solid) were obtained from Sigma-Aldrich (St Louis, MO). Sorbitol, aqueous H2O2 solution (31% w/v), and ammonium ferrous sulfate were obtained from Kanto Chemical (Tokyo, Japan). 21

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Fig. 5. Time-dependent changes in contents of products, subsrates, and H2O2 during enzymatic synthesis of theaflavins TF1 (A–D), TF2A (E–H), TF2B (I–L), and TF3 (M–P) by incubation of two selectively combined catechins with 0.1 mg/mL tyrosinase in the absence (●) or presence (△) of 0.2 mg/mL catalase. Data from incubation without any enzymes are shown as the control (○). Data are means ± SD (n = 3).

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Fig. 5. (continued)

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Fig. 6. Time-dependent changes in contents of TF2B and catechin substrates during incubation of mixtures of ECg and EGC at ratios of 1:1 (A–C), 1:2.4 (D–F), 1:3.6 (G–I), and 1:4.8 (J–L) with 0.1 mg/mL tyrosinase in the absence (●) or presence (△) of 0.2 mg/mL catalase. Data are means ± SD (n = 3). H2O2 in reaction media containing catalase (△ in D, H, L, and P) was not detected, so its concentration was indicated as 0 μM.

Xylenol orange and 4-methyl catechol were purchased from Wako Pure Chemical (Osaka, Japan). Purified theaflavins were prepared as described previously [24]. All other reagents used were of analytical grade.

citric acid to stop the reaction. Catechins and theaflavins in the stopped samples were analyzed on a 150 mm × 4.6 mm i.d., 4 μm, Synergi Polar-RP 80 Å column (Phenomenex, Torrance, CA) by RP-HPLC with eluting conditions of 0-10-30 min/16-20-40% MeCN solutions containing 0.05% phosphoric acid at flow rate of 1 mL/min and detected at 280 nm to identify the peaks of tea polyphenols. Each retention time (tR) of catechins and theaflavins was identified using the standards obtained as described above. Chromatogram analysis was performed with the data processing software Chromato-PRO (Run Time Corp., Tokyo, Japan).

2.2. Tyrosinase-catalyzed reaction with catechins and HPLC Respective catechin solutions were prepared by 10% ethanol/ 50 mM sodium phosphate buffer (pH 6.0) in the absence or presence 0.2 mg solid/mL catalase. Each catechin solution or selectively combined two catechin mixture (450 μL) was mixed with 50 μL of tyrosinase solution (1 mg solid/mL). The reaction mixture containing tyrosinase (at final concentration of 0.1 mg solid/mL) was incubated at 25 °C, shaken with a vortex mixer at intervals of 10 min or 15 min, and then each aliquot (50 μL) was taken and added into 300 μL of 25 mM

2.3. Measurement of H2O2 After incubation of catechin solutions in the presence or absence of tyrosinase, their H2O2 contents were measured spectrometrically using 24

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Fig. 6. (continued)

a ferrous ion oxidation (FOX) assay [25] with some modifications. Briefly, 50 μL of the reaction mixture was added into 300 μL of 25 mM citric acid, and the sample solution was passed through a C18H050 column (Toyo Roshi, Tokyo, Japan) to remove polyphenols that inhibit the FOX assay. The passed solution of 25 μL was reacted with 200 μL of FOX reagent, which is composed of 100 μM xylenol orange, 250 μM ammonium ferrous sulfate, 100 mM sorbitol, and 25 mM H2SO4, for 30 min at 30 °C. The absorbance of colored complex at 570 nm was measured by a Multiskan FC microplate reader (Thermo Fisher Scientific, Tokyo, Japan).

was mixed with 150 μL of 10 mM 4-methyl catechol dissolved in 50 mM sodium phosphate buffer (pH 6.0). The enzyme activity was determined at 25 °C using a Multiskan FC microplate reader at 414 nm. 3. Results and discussion 3.1. Generation of H2O2 during incubation of catechins in the absence or presence of tyrosinase After a 30-min incubation of the respective four catechins, EC, EGC, ECg, and EGCg, at initial concentration of 5 mM in phosphate buffer at pH 6.0 in the absence of tyrosinase, the H2O2 content significantly increased in the EGC and EGCg solutions compared to those before incubation (Fig. 2A). These results are consistent with previous reports indicating that phenolic compounds with a gallyl moiety show high H2O2-generating ability during their autooxidation [20–22]. Meanwhile, in the presence of tyrosinase from mushroom, catechins decreased (Fig. 2B) and the H2O2 content significantly increased in the EC,

2.4. Colorimetric assay of tyrosinase activity After 0.1 mg solid/mL tyrosinase was preincubated with H2O2 in 50 mM sodium phosphate buffer (pH 6.0) at 25 °C, an aliquot (50 μL) was added into an equal volume of catalase solution (0.2 mg solid/mL) to remove H2O2 in media. The residual tyrosinase activity was assessed by a colorimetric method. In this experiment, 15 μL of enzyme solution 25

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Fig. 7. Proposed schemes for H2O2-induced inhibition of tyrosinase-catalyzed TF2B synthesis from ECg and EGC (A) and ameliorative effects of exogenously added catalase into the reaction media (B). Arrow width is proportional to the qualitative importance of the reaction pathway kinetic.

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Table 1 Maximum yields of TF2B during a 60-min reaction as shown in Fig. 6. TF2B (mM) ECg : EGC 1 1 1 1

: : : :

1 2.4 3.6 4.8

(-)catalase 0.20 0.23 0.16 0.09

± ± ± ±

0.01 0.01 0.01 0.00

(+)catalase 0.24 0.31 0.34 0.28

± ± ± ±

*

0.00 0.02* 0.01* 0.04*

Based on consumed ECg (%)

Based on consumed EGC (%)

(-)catalase

(+)catalase

(-)catalase

(+)catalase

43.7 46.9 56.4 79.6

35.7 49.5 52.8 72.2

14.6 ± 0.9 7.9 ± 0.9 4.2 ± 0.3 3.0 ± 0.3

16.2 ± 0.7 11.9 ± 1.9 7.5 ± 0.5* 5.5 ± 0.6*

± ± ± ±

4.6 7.2 11.3 11.7

± ± ± ±

1.6 1.8 7.0 10.4

Data are means ± SD (n = 3). *Significantly different from (-)catalase (n = 3, P < 0.05).

EGC, and ECg solutions (Fig. 2A). For the EC and ECg solutions, in particular the latter, the extents of increase in the H2O2 content were greater in the presence of tyrosinase than in its absence. Thus, the tyrosinase-catalyzed oxidation of catechol-type catechins should be involved in H2O2 generation. The reason why tyrosinase generates more H2O2 when the substrate was ECg is unclear. The lack of correlation between the concentrations of H2O2 and the amounts of enzymatically consumed substrates (Fig. 2) might be due to differences in the phenolic structures that bind to the active site in an alternative orientation and contribute to suicide inactivation releasing H2O2 [17,18]. It is possible that ECg effectively binds to the active site and causes the suicide inactivation of tyrosinase, because ECg has the lowest KM value (0.8 mM) among the four catechins [10].

effects of catalase were observed in the synthesis of TF1 from EC and EGC (Fig. 5A–D), TF2A from EC and EGCg (Fig. 5E–H), and TF2B from ECg an EGC (Fig. 5I–L). The synthesis of TF3 from ECg and EGCg showed a lower yield than those of other four theaflavins (Fig. 5M), where the H2O2 content in media did not significantly increase even in the presence of tyrosinase (Fig. 5P). Thus, the low yield of TF3 should not be attributed to H2O2-induced enzyme inactivation. Considering slower consumption rates of EC and ECg in the reactions containing EGCg than EGC (Fig. 5B, F, J, N), as well as our previous study that indicated a remarkably slow synthesis of theaflavins from EGCg-rich green tea extract by tyrosinase [10], it is suggested that a higher level of EGCg could inhibit the tyrosinase-catalyzed oxidation of the other catechins competitively and/or concurrently reduced EC-and ECg-quinones. The yields of the other three theaflavins were still low based on the consumed pyrogallol-type catechins, however, this is due to both redox reactions between catechol quinones and gallyl moieties and the self-dimerization of pyrogallol-type catechins (Fig. 1) [10,27–29]. The reaction media containing EGC showed relatively high concentrations of H2O2 (Fig. 5D and L). One reason why H2O2 concentrations were lower in the presence of tyrosinase than in its absence might be that the rapid decrease of EGC restricted its autooxidation, which was responsible for the release of H2O2. Considering the time-dependent increase of H2O2 during the tyrosinase-catalyzed synthesis of TF2B (Fig. 5L), the elimination of H2O2 by catalase could be much more effective for enhancing this reaction. Furthermore, to elevate the rate of non-enzymatic condensation between the two quinones derived from ECg and EGC for the synthesis of TF2B, these two quinones should be concomitantly sufficient in the reaction media. In the case of a normal reaction, tyrosinase preferentially oxidizes ECg with about five times high kcat/KM as EGC [10]; however, the ECg quinone is reduced to ECg through a redox reaction with EGC, and the resulting EGC quinone causes self-dimerization [10,27]. EGC at high concentrations could react with tyrosinase competitively against ECg, increasing the condensation of their quinones. However, the more EGC is added, the more H2O2 is released by its autooxidation. Thus, the H2O2-induced inactivation of tyrosinase should be canceled by the addition of catalase. Tyrosinase was added into solutions containing ECg and EGC at different ratios, 1:1, 1:2.4, 1:3.6, and 1:4.8, with the initial concentration of ECg being low to suppress the suicide inactivation of tyrosinase, in the absence or presence of catalase (Fig. 6). We observed that TF2B continuously increased until almost all EGC was diminished, suggesting that the subsequent decrease in TF2B was due to its oxidative degradation by the ECg quinone, which accumulated in the media because the redox reaction with EGC no longer occurred. In the absence of catalase, the maximum yield of TF2B increased by elevating the initial ratio of EGC up to 2.4 (Fig. 6A and D); however, the decrease in ECg became slower by elevating the initial EGC/ECg ratio (Fig. 6B, E, H, and K), and the formation rate of TF2B decreased with the further increase of EGC (Fig. 6G and J). These results suggest that an excessive amount of EGC contributed to the redox reaction, reducing ECg quinone to ECg as well as the H2O2-induced inactivation of tyrosinase (Fig. 7A). The calculated low yields based on the consumed amount of EGC (Table 1) suggested that abundant EGC quinone in the reaction media also

3.2. Time- and dose-dependent inactivation of tyrosinase by H2O2 The H2O2 content increased up to about 40 μM during a 30-min incubation of 5 mM of the respective four catechins in the absence or presence of tyrosinase (Fig. 2A). To investigate effects of H2O2 around 40 μM on tyrosinase, its activity was measured by a colorimetric assay using 4-methyl catechol as a substrate. As a result, preincubation with H2O2 from 12.5 to 100 μM caused a time- and dose-dependent tyrosinase inactivation regardless of the subsequent removal of H2O2 by catalase (Fig. 3). This result suggests that H2O2 irreversibly inactivates tyrosinase interacting with the active site to which catalase could not access [19]. The tyrosinase activities estimated with the initial consumption rates for EC, EGC, ECg, and EGCg also decreased to 80%, 46%, 59%, and 62%, respectively, after the enzyme was pretreated with H2O2 (Fig. 4). The difference in sensitivity to H2O2 between catechins might be in part relevant to the highest kcat/KM value of tyrosinase for EC and the lowest kcat/KM and higher KM (5.55 mM) for EGC among the four catechins [10], i.e., EC, a preferable substrate to tyrosinase, could be rapidly oxidized by the enzyme which is still active form. Meanwhile, ECg with the second highest kcat/KM value [10] was relatively sensitive to H2O2 (Fig. 4). The tyrosinase-catalyzed oxidation of ECg, which significantly released H2O2 (Fig. 2A), showed a time-dependent decrease in the reaction rate [10] and it was influenced by co-incubation with neither H2O2 nor catalase (data not shown). These results suggest that, in the case of tyrosinase-catalyzed oxidation of ECg, H2O2 is generated near the active site and rapidly inactivates the enzyme. Taken together, tyrosinase is inactivated by H2O2 during its reaction with catechins, which is released through either autooxidation of EGC and EGCg or enzymatic oxidation of EC and ECg. 3.3. Effects of elimination of H2O2 on tyrosinase-catalyzed synthesis of theaflavins Theaflavins are synthesized by a tyrosinase-catalyzed reaction with selectively combined catechol and pyrogallol-type catechins [10,26]. However, under such conditions, H2O2 would accumulate in the reaction media, leading to enzyme inactivation. Therefore, we investigated whether the elimination of H2O2 by co-incubation with catalase enhances the tyrosinase-catalyzed synthesis of theaflavins. Ameliorative 27

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enhanced self-dimerization, probably forming theasinensins [27]. The increase in yield of TF2B based on consumed ECg with an elevated EGC/ECg ratio implied that ECg quinone-related oxidative degradation of TF2B might have been suppressed (Table 1). Meanwhile, in the presence of catalase, TF2B was more rapidly synthesized, and the consumption rates of both ECg and EGC were higher compared with those in the absence of the enzyme (Fig. 6), indicating that the H2O2induced inactivation of tyrosinase was reduced by catalase. In addition, the higher ratios of EGC/ECg may allow for the interaction of EGC with tyrosinase to overcome the higher kcat/KM for ECg, resulting in the effective formation of EGC quinones. Such conditions improved the efficiency of condensation between ECg- and EGC quinones, increasing the maximum yield of TF2B (Fig. 7B, Table 1). Catechin contents in tea leaves in general are known to be in the order of EGCg > EGC > ECg > EC, though there are differences between tea varieties, cultural conditions, and methods for quantitative analysis, including extraction [30,31]. ECg is too hydrophobic to be solubilized at high concentrations; in contrast, EGC is highly soluble in aqueous buffer solutions required for enzymatic reactions. Therefore, the use of a mixture of ECg and EGC at an appropriate ratio, with the latter being higher, should be feasible for more efficient synthesis of TF2B by tyrosinase with the assistance of catalase.

[8] [9]

[10] [11]

[12] [13]

[14] [15]

[16]

4. Conclusion H2O2 increased in the reaction media during incubation of each of the four catechins, EC, EGC, ECg, and EGCg, in the absence or presence of tyrosinase. H2O2 was significantly generated by autooxidation of EGC and enzymatic oxidation of ECg. Since a mixture of EGC and ECg is required for the tyrosinase-catalyzed synthesis of TF2B, H2O2 produced during the synthetic reaction should inactivate the enzyme. When H2O2 was eliminated by the addition of catalase into the tyrosinase-catalyzed reaction with ECg and EGC, the formation rates and yields of TF2B significantly increased. Furthermore, this study indicated that using EGC at an appropriately higher concentration than ECg is a promising strategy for improving the synthetic yield of TF2B in laboratory and industrial settings.

[17]

[18]

[19]

[20] [21]

Acknowledgements [22]

This work was supported by JSPS KAKENHI Grant Number JP17K07823, and a grant from the Cross-Ministerial Strategic Innovation Promotion Program (SIP), Urgent Project for Development and Diffusion of Innovative Technology towards Realization of the Aggressive Agriculture, Forestry, and Fisheries.

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