Specificity of tyrosinase-catalyzed synthesis of theaflavins

Article abstract of DOI:10.1016/j.molcatb.2017.03.009

This study kinetically characterized the mechanism of the enzymatic synthesis of theaflavins (TFs) from catechins by mushroom tyrosinase (EC 1.14.18.1). In reactions containing one of four catechins, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), and their galloylated forms (ECg and EGCg), they were oxidized by tyrosinase with apparent K_M values of 3.78, 5.55, 0.80, and 3.05 mM, respectively, and with different consumption rates, of which EC was more than four times higher than those of the others. In reactions with binary combinations of catechins with tyrosinase, the synthesis of TF1 from EC and EGC occurred most efficiently, while the yields of mono- and di-galloylated TFs, TF2A, TF2B, and TF3, were low. Time-dependent changes in concentrations of the reactants suggested that the enzymatic oxidation of catechins and the subsequent non-enzymatic coupling redox reaction between the quinone derived from EC or ECg and the intact pyrogallol-type catechin (EGC or EGCg) proceeded concurrently. The latter reaction induced the rapid decrease of EGC and EGCg and it was remarkable for EGCg. So the efficiency of condensation of a pair of quinones from catechol- and pyrogallol-type catechins is restricted, critically influencing the yield of TFs. Using green tea extracts as mixtures of the four substrate catechins, tyrosinase produced TF1 most abundantly. Furthermore, a remarkable enhancement of production of TF2A and TF2B as well as TF1 was observed, when the initial concentration of EGCg was low. These results suggest that the catechin composition has an impact on yields of TFs.

Full text of DOI:10.1016/j.molcatb.2017.03.009

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Specificity of tyrosinase-catalyzed synthesis of theaflavins
∗
Asako Narai-Kanayama , Aya Kawashima, Yuuka Uchida, Miho Kawamura,
Tsutomu Nakayama
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
a r t i c l e i n f o
a b s t r a c t
Article history:
This study kinetically characterized the mechanism of the enzymatic synthesis of theaflavins (TFs) from
catechins by mushroom tyrosinase (EC 1.14.18.1). In reactions containing one of four catechins, (-)-
epicatechin (EC), (-)-epigallocatechin (EGC), and their galloylated forms (ECg and EGCg), they were
oxidized by tyrosinase with apparent KM values of 3.78, 5.55, 0.80, and 3.05 mM, respectively, and with
different consumption rates, of which EC was more than four times higher than those of the others. In
reactions with binary combinations of catechins with tyrosinase, the synthesis of TF1 from EC and EGC
occurred most efficiently, while the yields of mono- and di-galloylated TFs, TF2A, TF2B, and TF3, were
low. Time-dependent changes in concentrations of the reactants suggested that the enzymatic oxidation
of catechins and the subsequent non-enzymatic coupling redox reaction between the quinone derived
from EC or ECg and the intact pyrogallol-type catechin (EGC or EGCg) proceeded concurrently. The latter
reaction induced the rapid decrease of EGC and EGCg and it was remarkable for EGCg. So the efficiency
of condensation of a pair of quinones from catechol- and pyrogallol-type catechins is restricted, critically
influencing the yield of TFs. Using green tea extracts as mixtures of the four substrate catechins, tyrosi-
nase produced TF1 most abundantly. Furthermore, a remarkable enhancement of production of TF2A and
TF2B as well as TF1 was observed, when the initial concentration of EGCg was low. These results suggest
that the catechin composition has an impact on yields of TFs.
Received 13 December 2016
Received in revised form 15 March 2017
Accepted 23 March 2017
Available online xxx
Keywords:
Tyrosinase
Catechin
Theaflavin
Enzymatic synthesis
©
2017 Elsevier B.V. All rights reserved.
1
. Introduction
as well as their application into foods and pharmaceutical products
has been restricted.
Theaflavins (TFs), the characteristic pigments in black tea leaves
TFs are known to be produced during the process of fermen-
tation of the leaves of Camellia sinensis, in which endogenous
polyphenol oxidase (PPO) is involved. Condensation of two
oxidized catechins and decarboxylation afford TF production
[14,15]. Condensation of different pairs of catechins, one with
a di-hydroxylated B-ring (catechol-type) and the other with
a tri-hydroxylated B-ring (pyrogallol-type), results in forma-
tion of four kinds of TFs, theaflavin (TF1), theaflavin-3-O-gallate
(TF2A), theaflavin-3 -O-gallate (TF2B), and theaflavin-3,3 -O-
digallate (TF3) (Fig. 1). TF synthesis by plant PPOs has been
investigated [14,15], and a possible mechanism has been proposed
as shown in Fig. 1. Some reasons why the yield of TFs is generally
low have been suggested as follows: 1) the plant PPOs preferentially
catalyze catechols rather than pyrogallols [16,17], 2) pyrogallols
are susceptible to oxidation by quinone, reducing it to catechol, 3)
the quinone, an oxidative product of catechin, is so highly reactive
that it binds to other molecules possessing nucleophilic amino- and
thiol groups, such as proteins [18], and it also attacks TFs leading to
their degradation [15,19] and 4) further uncontrolled reactions pro-
and their exudates, are known to contribute to the orange or
orange-red color and astringency of various kinds of brewed tea.
Recently, TFs have attracted attention due to their various bioac-
tivities, such as antioxidant activity against LDL oxidation [1],
radical-scavenging activity [2], anticancer activity [3,4], antidia-
betic effects [5], and inhibitory activities against the cholesterol
and fat absorption [6–8] and the bone loss in models of osteoporo-
sis [9]. However, the amount of TFs in black tea is fairly low, about
ꢀ
ꢀ
8
–20 mg/100 mL tea brewed from a teabag [10]. In addition, TFs are
not stable in aqueous solution, especially under neutral or alkaline
conditions [11–13]. Therefore, research on the bioactivities of TFs
Abbreviations: EA, ethyl acetate; EC, (-)-epicatechin; EGC, (-)-epigallocatechin;
ECg, (-)-epicatechin gallate; EGCg, (-)-epigallocatechin gallate; GLB, green tea leaves
etracted with the buffer; GPE, green tea powder extracted with ethyl acetate; PPO,
polyphenol oxidase; TF, theaflavin.
∗ Corresponding author. Tel.: +81 422 31 4151; fax: +81 422 51 9984.
E-mail address: a-narai@nvlu.ac.jp (A. Narai-Kanayama).
http://dx.doi.org/10.1016/j.molcatb.2017.03.009
1
381-1177/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: A. Narai-Kanayama, et al., Specificity of tyrosinase-catalyzed synthesis of theaflavins, J. Mol. Catal. B:
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Fig. 1. Structures of tea polyphenols and prospective mechanism of polyphenol oxidase- catalyzed synthesis of theaflavins from catechins.
ceed providing polyphenol oligomers, for example, theasinensins,
theanaphtoquinones, dehydrotheaflavins, bistheaflavins, and so on
der (AVT Natural Products Ltd., Chennai, India) was provided by Tea
Solutions, Hara Office Inc. (Tokyo, Japan) and green tea leaves were
commercially obtained. All other reagents used were of analytical
grade.
[
20]. In addition, in vitro reactions using PPO and peroxidase (POD)
purified from tea leaves have recently demonstrated that once
synthesized TFs are converted to the reddish-brown thearubigins,
which are detected as a broad hump on RP-HPLC chromatograms,
probably through the POD-catalyzed reaction [21,22]. Since the
enzymatic and non-enzymatic reactions proceed concurrently and
their sub-products interfere with the synthesis of TFs, it is difficult
to understand the overall reaction mechanisms.
2.2. Tyrosinase-catalyzed oxidation of catechins
Unless otherwise specified, enzymatic reactions were per-
formed using tyrosinase at 0.05 mg/mL (156 mU/mL) with each
catechin and 0.1 mg/mL (313 mU/mL) for TF synthesis in 50 mM
Tyrosinase (EC 1.14.18.1) from the mushroom Agaricus bis-
◦
Na-phosphate buffer, pH 6.0, at 25 C without pH control.
porus,
a copper-containing oxidase with both monophenol
Respective catechin solutions (10 mM) were prepared using 20%
ethanol/40 mM Na-phosphate buffer, pH 6.0. After certain incu-
bation periods, an aliquot (100 ␮L) of the reaction mixture was
collected and added into 1 mL of 25 mM citric acid solution (pH 2.4)
to stop the reaction. These samples were cooled in an autosam-
monooxygenase- and diphenol oxidase activities, has been char-
acterized and used in various studies [23–27]. This mushroom
tyrosinase is able to oxidize pyrogallols in addition to catechols
[
28]. If it reacts with catechol- and pyrogallol-type catechins con-
currently producing both-types of quinones, condensation of these
two oxidative products could be accelerated, increasing TF produc-
tion. Thus, tyrosinase may be a promising enzyme as a catalyst for
the efficient synthesis of TFs. Indeed, it has been used for in vitro
TF synthesis from catechins in place of PPO [29,30]. However, to
our knowledge, the optimum reaction conditions and substrate
specificities as well as TF yields have not been fully investigated
yet. In this study, we compared the tyrosinase-catalyzed reactions
not only with single catechin but also with binary combinations of
catechins. We found that the enzymatic specificities for catechins
were not directly responsible for the yields of the corresponding
TFs. Furthermore, the addition of tyrosinase into green tea extracts
differing in catechin composition indicated that TF1 was preferen-
tially produced and that a higher concentration of EGCg suppressed
tyrosinase-catalyzed TF synthesis.
◦
pler (L-2200, Hitachi, Tokyo) at 5–8 C and analyzed by RP-HPLC,
which was carried out using a Hitachi HPLC system (L-2130 pump,
L-2400 UV detector) equipped with a SUS line filter (GL Science,
TM
Tokyo, Japan) and a Phenomenex Synergi
4.6 mm × 150 mm) column (Shimadzu GLC, Tokyo, Japan). Cate-
chins were eluted using an aqueous 20% MeCN solution containing
4 ␮m Polar-RP 80 A
˚
(
0
2
.05% phosphoric acid at flow rate of 1 mL/min, and detected at
80 nm. For TF1, TF2A, TF2B, and TF3, aqueous 32% MeCN solu-
tions containing 0.05% phosphoric acid were used as eluents, and
each retention time (tR) was identified using the TF standards,
which were obtained as described previously [31]. The analysis of
chromatograms was performed with the data processing software
Chromato-PRO (Run Time Corporation, Tokyo, Japan).
2.3. Preparation of catechin mixtures from green tea
2
. Materials and methods
In order to investigate the effects of the differences of catechin
composition on the product yields in tyrosinase-catalyzed TF syn-
thesis, green tea powder and a type of green tea leaf were used
for the extraction of catechins. For the green tea powder, 3 mL of
0.1 M citric acid solution was added to 170 mg of powder. After agi-
2
.1. Materials
EC, EGC, ECg, and EGCg were kindly provided by Mitsui Norin Co.,
◦
Ltd. (Shizuoka, Japan). Tyrosinase from mushroom was obtained
from Sigma-Aldrich Co. (St Louis, MO, USA). Instant green tea pow-
tation for 3 min, it was centrifuged at 8400 × g for 15 min at 25 C.
Its supernatant was mixed well with a 1.5-fold volume of ethyl
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Fig. 2. Kinetic analysis of the tyrosinase-catalyzed reaction with each catechin.
Each of four catechins, EC (open circle), EGC (open triangle), ECg (closed circle), and EGCg (closed triangle), was reacted with 0.05 mg/mL tyrosinase in 50 mM Na-phosphate
◦
buffer, pH 6.0, for 3 min at 25 C. Data of consumption rates are indicated as means ± SD (n = 3). Apparent values of KM (mM) and Vmax (mM/min) are also described.
acetate (EA), and then left to stand for about 10 min. The upper
fraction of 2.4 mL was collected and evaporated. The concentrated
mixture was solubilized in 4.8 mL of 50 mM Na-phosphate buffer,
pH 6.0, just before use as the green tea powder extracted with EA
(
GPE) sample. For green tea leaves, 390 mg of leaves were infused
Scheme 1. Tyrosinase-catalyzed and quinone-induced oxidation of catechins.
Meanings of the symbols are as follows: S: catechin substrate, E: enzyme, ES:
enzymesubstrate complex, Q: quinone produced by enzymatic reaction with
substrate, D: dimer of substrate, DQ: dimer quinone, P: polymer produced by non-
enzymatic reaction related to quinone, k and k’: rate constants corresponding to
quinoneinduced chemical reactions.
in 6 mL of 50 mM Na-phosphate buffer, pH 6.0, at room temper-
ature for 5 min. After the infusion was agitated and centrifuged
similarly to the green tea powder, the supernatant was collected
and used within a day as the green tea leaves extracted with buffer
(
GLB) sample for the tyrosinase-catalyzed reaction. Each sample
of the two green tea extracts, GPE and GLB, were reacted with
tyrosinase at 25 C, and then diluted by an eight-fold volume of
◦
tions converting substrates into self-dimer or polymer, of which
rate constants are likely to be very high [32,33] (Scheme 1).
The direct measurement of substrate cannot give accurate
kinetic constants, such as KM, because the quinone is highly reac-
tive and unstable, resulting in large k and k’ [32]. However, when
the enzymatic oxidation of substrate was very slow together with
2
5 mM citric acid solution to stop the reactions. Aliquots of 50 ␮L
were analyzed by RP-HPLC which was carried out as described
above, except for the eluting conditions: 0–10–40–42–47 min,
aqueous 8–20–32–80–80% MeCN solution containing 0.05% phos-
phoric acid.
[
S] > > [Q], this rate-limiting step would realize [Q] ≈ 0 in the early
stage of reaction. Under such a condition, the rate of substrate con-
sumption could be expressed as follows:
3
. Results and discussion
dS
dt
ꢀ
3.1. Tyrosinase-catalyzed reaction with respective catechins
=
Venz + k[Q][S] − k [Q][D] ≈ Venz,
(1)
Since the optimum pH for tyrosinase from A. bisporus is around
where Venz = Vmax[S]/(KM + [S]). So we performed the direct anal-
6
–7 [23,28] and the stabilities of catachins and TFs are low under
yses of each of the four catechins incubated for a short-term
reaction (for 3 min) with 0.05 mg/mL of tyrosinase. The consump-
tion rates of catechins showed Michaelis-Menten saturation curves
(Fig. 2). We confirmed the proportional relationship between
|dS/dt| and [E] at below 0.05 mg/mL (data not shown), which sug-
gested the condition permitting Eq. (1). Therefore, the apparent KM
and Vmax values for EC, EGC, ECg, and EGCg, as indicated in Fig. 2,
were tentatively determined by Lineweaver-Burk plots. The differ-
neutral and alkaline conditions [11–13], the reaction was per-
formed at pH 6.0 in this study. We confirmed that EC, EGC,
ECg, EGCg, and TFs were relatively stable and almost all of them
remained during a 60-min incubation in the absence of tyrosi-
nase in 50 mM Na-phosphate buffer, pH 6.0, at 25 C (data not
shown). During the tyrosinase-catalyzed reaction with catechins,
the quinones derived from substrates cause non-enzymatic oxida-
◦
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ences betweenvalues of the apparentKM in this study and the Kppo
M
determined by Munoz-Munoz et al. [33] might be due to the differ-
ences in assays as well as reaction pHs. However, the highest affinity
of tyrosinase for ECg is agreement with their result determined by
the chronometric method based on a spectrophotometric measure-
ment [33]. Furthermore, the order of Vmax/KM of four catechins,
EC > > ECg > EGCg ≈ EGC, is also similar to that reported in the liter-
ature [33]. Since the galloylated catechins (ECg, EGCg) are known
to bind to proteins with higher affinity than non-galloylated ones
(
EC, EGC) [34–37], the former could also bind to tyrosinase non-
specifically via hydrophilic and hydrophobic interactions, making
it possible for the enzyme to accommodate them nearby its active
site.
To investigate effects of the subsequent non-enzymatic oxida-
tion of catechins on the tyrosinase-catalyzed reaction, 5 mM of each
catechin and 0.1 mg/mL tyrosinase were incubated for 60 min. The
order of overall substrate consumption was EC > EGCg > EGC > ECg
(
Fig. 3), which was inconsistent with the results shown in Fig. 2. This
Fig. 3. Time-dependent changes of catechins during the tyrosinase-catalyzed reac-
tion with respective catechins.
suggests that the pyrogallol-type catechins, in particular EGCg, are
highly reactive with their quinones possibly providing self-dimers
and polymers effectively. For ECg, in spite of the lowest KM, its con-
sumption became slower after 30 min (Fig. 3). This might be due to
the product inhibition, on which further studies are undertaken.
Each of four catechins, EC (open circle), EGC (open triangle), ECg (closed circle), and
EGCg (closed triangle), at 5 mM was reacted with 0.1 mg/mL tyrosinase in 50 mM
◦
Na-phosphate buffer, pH 6.0, for 60 min at 25 C. Data are indicated as means ± SD
(
n = 3).
3
.2. Tyrosinase-catalyzed reaction with binary combinations of
(Fig. 4A − D), suggesting that the former catechins were oxidized
by not only tyrosinase but also quinones derived from the latter
ones. As tyrosinase preferentially reacted with EC about 5.8-times
faster than ECg (Fig. 2), the rates of the pyrogallol oxidation by
EC-quinone (Fig. 4A&B) seemed to be higher than those by ECg-
quinone (Fig. 4C&D). Meanwhile, the consumption rates of EC and
ECg were suppressed especially at the early stage of the reaction
catechins
In the tyrosinase-catalyzed reactions with different binary com-
binations of 2 mM catechins, the respective reactions produced the
corresponding TFs (Fig. 4A − D). Pyrogallol-type catechins (EGC,
EGCg) were consumed faster than catechol-type ones (EC, ECg)
Fig. 4. Time-dependent changes of catechins and TFs during the tyrosinase-catalyzed synthesis of TF1 (A), TF2A (B), TF2B (C), and TF3 (D).
Initial substrate concentrations were 2 mM. Temporal changes in concentrations of EC (open circles), EGC (open triangles), ECg (closed circles), EGCg (closed triangles), and
◦
corresponding TFs (closed red squares) are indicated. Enzymatic reaction was conducted with 0.1 mg/mL tyrosinase in 50 mM Na-phosphate buffer, pH 6.0, at 25 C. Data are
indicated as means ± SD (n = 3).
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Table 1
Maximum yields of TFs in tyrosinase-catalyzed synthetic reactions (Fig. 3). Each reaction time (min) when we observed the highest TF yield is shown.
TF synthesis from catechins
min
TF yields (%) based on consumed catechins with different B-ring types
Catechol (EC or ECg)
Pyrogallol (EGC or EGCg)
TF1
2 mM EC: 2 mM EGC
2 mM EC: 2 mM EGCg
2 mM ECg: 2 mM EGC
2 mM ECg: 2 mM EGCg
20
20
30
30
75.3 ± 4.7
65.3 ± 4.0
48.9 ± 3.4
13.9 ± 0.7
34.5 ± 3.5
12.4 ± 0.9
19.2 ± 1.2
4.8 ± 0.1
TF2A
TF2B
TF3
Data are indicated as means ± SD (n = 3).
combined with EGCg (Fig. 4B&D) more than EGC (Fig. 4A&C). This
represents that EGCg is more susceptible to the quinone-induced
oxidation of pyrogallol than EGC. Although the molecular mobil-
ity of EGCg may be also restricted by its non-specific binding to
tyrosinase, this property would allow EGCg to contact effectively
with quinones released from the catalyst. Taking into account the
susceptibility of EGCg against ECg- and self-quinones as described
above, the non-enzymatic condensation between ECg- and EGCg-
quinones rarely occurred, resulting in the slow production of TF3
(Fig. 4D). The faster production of TF1 than TF2A also reflected the
lower susceptibility of EGC than EGCg (Fig. 4A&B).
TFs continued to increase until the complete consumption of
pyrogallols, and later, markedly decreased (Fig. 4). Since we con-
firmed that TFs were not directly consumed by tyrosinase (data
not shown), quinones and/or other products synthesized by the
tyrosinase-catalyzed oxidation of catechols probably caused elec-
trophilic attacks on TFs, leading to their breakdown [15,20]. High
amounts of EGC and EGCg in the reaction mixture could inhibit
the quinone-induced TF breakdown through the coupled reduc-
tion of catechol quinones with their pyrogallol groups. However,
even if the pyrogallols remained, both synthesis and breakdown of
TFs would occur concurrently in the reaction. The ratio between
catechol- and pyrogallol-catechins is therefore an important factor
for the yield of TFs.
TF yields based on the consumed pyrogallol-type catechins
were lower than those based on catechol-type catechins (Table 1).
EGC and EGCg were susceptible to the quinone-induced oxidation,
reducing quinones to the original catechols. The resulting EGC-
and EGCg-quinones were likely to provide dehydrotheasinensins
through the subsequent non-enzymatic coupling reaction [38,39].
A comparison of maximum TF yields (Table 1) indicated that TF1
was most efficiently synthesized. In contrast, the yield of TF3 was
extremely low. For example, ratios of the TF yields were as fol-
lows: TF2A/TF1 was 0.87 on the EC base (reflecting the effect of
substrate exchanging EGC for EGCg), TF3/TF2B was 0.28 on the ECg
base (exchanging EGC for EGCg), TF2B/TF1 was 0.56 on the EGC
base (exchanging EC for ECg), and TF3/TF2A was 0.39 on the EGCg
base (exchanging EC for ECg). These differences between TF3 and
the others seemed to be beyond the properties of the tyrosinase-
catalyzed catechin oxidation as shown in Figs. 2 and 3. Thus,
further studies are currently undertaken to investigate whether the
lower efficiency of condensation between ECg- and EGCg-quinones
resulted from a certain regioselective control or not.
Fig. 5. Chromatograms of green tea extracts (upper: GPE, lower: GLB).
Table 2
Catechin compositions in green tea extracts.
(
mM)
EC
EGC
ECg
EGCg
GPE
GLB
1.2
1.0
2.7
3.9
0.95
0.12
4.3
0.86
Table 3
Concentrations of TFs in reaction mixtures after a 48-min reaction with 0.1 mg/mL
◦
tyrosinase at 25 C.
(
␮M)
TF1
TF2A
TF2B
TF3
GPE
GLB
42 ± 1.5
6.6 ± 0.8
21 ± 1.6
7.8 ± 0.7
30 ± 1.4
5.9 ± 0.7
2.1 ± 0.3
174 ± 8.9
Data are indicated as means ± SD (n = 3).
caffeine, 50% EGCg, and 60% ECg, in order of retention time in RP-
HPLC (Fig. 5). Although hydrophilic EGC was lost to a remarkable
extent through EA extraction, other catechins were more effec-
tively extracted. The twice-diluted green tea powder-EA extract
was used in this study as a GPE sample. On the other hand, the
green tea leaves were immersed and agitated in phosphate buffer,
pH 6.0, giving the green tea leaves-buffer extract (GLB). The overall
extraction efficiency of the leaves was low compared to the pow-
der, however, hydrophilic EGC was more abundant than EGCg in
GLB (Fig. 5, Table 2).
3.3. Tyrosinase-catalyzed TF synthesis using green tea extracts
We prepared two green tea extracts to investigate the
tyrosinase-catalyzed TF synthesis from available catechin mixtures.
One was extracted from a commercially obtained instant green tea
powder with an acidic solution, which was used to prevent catechin
degradation. As the fine powder provided a green solution rich in
not only catechins but also other water-soluble components such
as caffeine, amino acids, and proteins (data not shown), catechins
were subsequently extracted from the solution with ethyl acetate
◦
During the incubation of GPE or GLB with tyrosinase at 25 C,
TFs increased linearly over time for 48 min (data not shown). TF1
was predominantly synthesized in both samples (Fig. 6, Table 3).
TF3 was less produced in the GLB sample (Fig. 6B, Table 3) due to
lower concentrations of both ECg and EGCg (Table 2). The GPE sam-
ple showed slow TF production (Fig. 6A), probably because a large
(
EA). The EA fraction recovered about 25% EGC, 40–50% EC, 30%
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cific bioactivities [6,7,40,41], their selective syntheses are desired
for their use in in vitro and in vivo studies, and their application
to foods, beverages, and pharmaceutical products. Further investi-
gation focusing on the effects of catechin composition on TF yields
would suggest the proper conditions for tyrosinase-catalyzed TF
synthesis. When using available green tea leaves as a source of
catechins, the catechin composition could be customized by the
extraction procedures, such as by the solvents used.
Acknowledgments
This work was supported by 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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Enzym. (2017), http://dx.doi.org/10.1016/j.molcatb.2017.03.009

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Enzym. (2017), http://dx.doi.org/10.1016/j.molcatb.2017.03.009