Exploring the catalytic mechanism of a novel β-glucosidase BGL0224 from Oenococcus oeni SD-2a: Kinetics, spectroscopic and molecular simulation

Article abstract of DOI:10.1016/j.enzmictec.2021.109814

The β-glucosidase derived from microorganisms has attracted worldwide interest for their industrial applications, but studies on β-glucosidases from Oenococcus oeni are rare. In this paper, catalytic mechanism of a novel β-glucosidase BGL0224 of Oenococcu

Full text of DOI:10.1016/j.enzmictec.2021.109814

Enzyme and Microbial Technology 148 (2021) 109814
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Enzyme and Microbial Technology
journal homepage: www.elsevier.com/locate/enzmictec
Exploring the catalytic mechanism of a novel β-glucosidase BGL0224 from
Oenococcus oeni SD-2a: Kinetics, spectroscopic and molecular simulation
Jie Zhang, Ning Zhao, Junnan Xu, Yiman Qi, Xinyuan Wei, Mingtao Fan*
College of Food Science and Engineering, Northwest A & F University, Yangling, Shaanxi, 712100, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The β-glucosidase derived from microorganisms has attracted worldwide interest for their industrial applications,
but studies on β-glucosidases from Oenococcus oeni are rare. In this paper, catalytic mechanism of a novel
β-glucosidase BGL0224 of Oenococcus oeni SD-2a was explored for the first time by kinetic parameters deter-
mination, fluorescence spectroscopy and quenching mechanism analysis, molecular dynamics simulation. The
results indicated that BGL0224 had universal catalytic effect on different types of glycoside substrates, but the
catalytic efficiencies were different. Fluorescence quenching analysis results suggested that the quenching pro-
cesses between BGL0224 and seven kinds of substrates were predominated by the static quenching mechanism. A
reasonable three-dimensional model of BGL0224 was obtained using the crystal structure of E.coli BglA as a
template. The analysis results of molecular simulation (RMSD, Rg, RMSF and hydrogen bonding) showed that the
composite system ‘BGL0224-pNPG’ was very stable after 40 ns. The catalytic process of BGL0224 acting on ‘p-
Nitrophenyl β-D-glucopyranoside’ conformed to the double displacement mechanism. Two glutamic acid residues
‘Glu178 and Glu377’ played a vital role in the whole catalytic process. Overall, this study gave specific insights
on the catalytic mechanism of BGL0224, which was of great significance for developing its potential applications
in food industry.
Amino acid
β-Glucosidase
Catalysis
Molecular docking
p-NPG
Food industry
1. Introduction
can promote biomass conversion. A thermotolerant β-glucosidase pro-
duced by the fungal strain BCC2871 (Periconia sp.) was found to possess
As a kind of cellulase, β-glucosidase (EC3.2.1.21) is widely found in
nature and it has been known for over 180 years since the first
description of the action of emulsion (almond β-glucosidase) on amyg-
dalin by Liebig and Wohler in 1837 [1]. Later researches indicated that
β-glucosidase could be derived from plants, animals and microorgan-
isms. The structure and enzymatic properties of β-glucosidase from
different sources vary widely. The main function of β-glucosidase is to
hydrolyze glycosidic bonds to release β-D-glucosyl residues [2]. There
are two main classification methods for β-glucosidases [3,4]. Based on
the amino acid sequences, β-glucosidases are classified as GH1, GH3,
GH5, GH9, GH30 and GH116 families [5]. The other is based on sub-
strate specificity. β-Glucosidases are categorized as aryl β-glucosidases,
true cellobiases and wide range substrate specificity enzymes [6].
Among various β-glucosidase, microbial-derived β-glucosidase is
attracting more industrial applications due to its advantages of
large-scale preparation, inexpensive, and environment-friendly [7].
β-Glucosidase is vital for many biological processes. For example, it
the ability of hydrolyzing rice straw into simple sugars [8]. β-Glucosi-
dase is also involved in the degradation of various functional glycoside
precursors (e.g., terpenols, flavonoids and phytohormones) and poten-
tially harmful metabolites (e.g., glycosylceramides) [9]. In addition,
β-glucosidase is regarded as a versatile industrial biocatalyst. Compared
to other industrial catalysts, β-glucosidase is significantly more stable
and avoid enzyme subunit desorption [10]. Furthermore, β-glucosidase
can hydrolyze many kinds of glycosides such as isoflavone glycosides
and pyridoxine glucoside, which is very important to enhance the aroma
profiles and quality of food products [11].
It is beneficial to study the catalytic mechanism of β-glucosidase and
to improve the catalytic efficiency of cellulose hydrolysis. In recent
years, many researchers were keen on exploring the molecular biology
of β-glucosidase. For instance, Yang [12] revealed the distinct catalytic
specificity and reaction kinetics of β-glucosidase (TN0602) through a
combination of enzyme-substrate thermodynamic binding and molecu-
lar docking simulation. The three-dimensional model of the barley
* Corresponding author.
E-mail address: fanmt@nwsuaf.edu.cn (M. Fan).
https://doi.org/10.1016/j.enzmictec.2021.109814
Received 17 February 2021; Received in revised form 14 April 2021; Accepted 29 April 2021
Available online 3 May 2021
0141-0229/© 2021 Elsevier Inc. All rights reserved.

J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
β-glucosidase gave an explanation not only for the enzyme’s ability to
hydrolyze some kinds of small, dimeric substrates but also for its pref-
erence for the relatively straight (1,4)-b-oligo glucoside substrates [13].
Pang [14] compared structures of the glucose-tolerant β-glucosidase
‘Bgl6’ and its variants at high resolution, which provided evidence for its
glucose-tolerant mechanism and thermostability. From these reports we
can see that detailed kinetic investigations and molecular simulation of
essential residues have been much important for functional annotation
of enzymes.
420 nm. One unit of β-glucosidase activity was defined as the amount of
enzyme that produces 1 μM of p-NP per min. β-Glucosidase activity was
calculated according to the standard curve of p-NP (Y = 0.0049X +
0.0185, R2 = 0.9997).
2.4. Effect of temperature and pH on enzyme activity
The purified β-glucosidase BGL0224 was dissolved in 20 mM sodium
phosphate buffer (pH 5.0) to a final concentration of 10 mg/mL. 10 μL of
As a patented strain, Oenococcus oeni SD-2a plays a significant role in
the process of wine fermentation, the most important reason is its high
β-glucosidase activity. On the one hand, β-glucosidase can hydrolyze
terpene glycosides and increase the aroma of wine. On the other hand, it
has been proved to be related to the decomposition of anthocyanin
glucosides, resulting color loss of wine in malolactic fermentation [15,
16]. In all, studies on β-glucosidases from Oenococcus oeni focused more
on the preliminary characterization and there were few reports on the
catalytic mechanism. Therefore, in-depth study of the β-glucosidase of
O. oeni SD-2a is necessary and of great significance. In our previous
research, we have obtained a novel β-glucosidase BGL0224 of Oeno-
coccus oeni SD-2a and its enzymatic properties were successfully char-
acterized [17]. In this study, basic kinetics, spectroscopic and molecular
simulation methods were applied to further understand the fundamental
structure–function relationships and elucidate its catalytic mechanism,
which is of great significance to develop its potential/performance as a
commercial enzyme.
the above solution was added to 490
substrates respectively. Then, 500 L of the mixture was incubated at a
temperature range of 25～70℃ for 30 min. 500 L of 1 M Na2CO3 was
μL solutions (25 mM) of seven
μ
μ
added to the mixture to terminate the reaction. The β-glucosidase ac-
tivities were measured according to the aforementioned method. The
effects of pH on different substrates were determined at the optimal
reaction temperature for each substrate. The purified enzyme BGL0224
was dissolved in 20 mM sodium phosphate buffer (pH 5.0) to a final
concentration of 10 mg/mL. Simultaneously, seven substrates were
dissolved in 20 mM sodium phosphate buffer with a pH range of 2.5～
7.5. 10
strates solution (25 mM) with different pH respectively. Then, 500
each mixture was incubated at the optimal reaction temperature for
μL of the enzyme solution was added to 490
μ
L of seven sub-
μ
L of
each substrate for 30 min. After that, 500 μL of 1 M Na2CO3 was added to
each mixture to terminate the reaction. The β-glucosidase activities were
measured according to the aforementioned method.
2.5. Determination of kinetic parameters of BGL0224
2. Materials and methods
Enzyme kinetics parameters of BGL0224 acting on seven kinds of
substrates were determined at the optimal reaction temperature and pH
2.1. Chemicals
for each substrate. 100 μg of purified enzyme BGL0224 was added to
All of the substrates with purity ≥99 % used in this study (p-Nitro-
phenyl β-D-glucopyranoside, p-Nitrophenyl β-D-galactopyranoside, p-
Nitrophenyl β-D-xylopyranoside, p-Nitrophenyl β-D-cellobioside, p-
various concentrations (0.1～25 mM) of each substrate. 500 μL of 1 M
Na2CO3 was added to each mixture to terminate the reaction at 5 min.
The Michaelis-Menten constant (Km) and maximum rate of the reaction
(Vmax) were measured using nonlinear fitting of the Michaelis-Menten
equation. Substrate concentrations were on the horizontal axis and the
reaction rates were on the vertical axis. The‘Hill’function (n = 1) in the
OriginPro 8.0 software was used to fit the Michaelis-Menten equation.
The turnover constant (Kcat) and the catalytic coefficient (ratio of Kcat to
Km) were then calculated.
Nitrophenyl β-D-glucuronide, p-Nitrophenyl
Nitrophenyl
α-D-glucopyranoside, p-
α
-D-galactopyranoside) were purchased from Yuanye Bio-
Tech, Co., Ltd (Shanghai, China). Chemical structures of seven kinds
of substrates were shown in Fig. S1. The remaining chemicals with pu-
rity ≥98 % were purchased from Solarbio Life-Science Co., Ltd (Beijing,
China), which mainly included sodium chloride (NaCl), sodium car-
bonate (Na2CO3), potassium phosphate monobasic (KH2PO4) and
disodium phosphate (Na2HPO4). Water was purified by a Milli-Q Direct
Water Purification System (Millipore, Billerica, MA).
2.6. Fluorescence spectra assays
Fluorescence evaluation was performed using a fluorescence spec-
trophotometer (PerkinElmer LS-55, Massachusetts, USA). The emission
and excitation wavelengths of this assay were at 390～520 and 340 nm,
respectively. The emission spectrum was measured from 390 to 520 nm
2.2. Preparation of β-glucosidase BGL0224
O. oeni SD-2a was obtained from a Chinese wine region in Shandong
province. The purified β-glucosidase BGL0224 belongs to glycoside hy-
drolase 1 (GH1) family and was expressed in our previous research [17].
Briefly, recombinant plasmid PcoldI-0224 was constructed and trans-
formed into E. coli BL21 (DE3) for expression. Then, the crude enzyme
solution was passed through a ‘His⋅tag Ni-NTA Superflow Column’
eluted with 300 mM imidazole and an ultrafiltration tube (30 kDa) to
obtain the purified enzyme. The apparent molecular weight of BGL0224
is 55.15 kDa.
in the presence of various concentrations (0～20 μM) of seven substrates
by using 2.5 and 9 nm slit for excitation at the temperatures of 303 K. All
measurements were carried out at enzyme concentration of 0.1 mg/mL
and temperature of 303 K.
2.7. Homology modeling
The amino acid sequence of BGL0224 (GenBank accession number:
MT330371) contained 480 amino acids. A BLAST search was performed
against PDB (Protein Databank) using target sequence as a query to get
the corresponding template for the protein BGL0224. For identification
of suitable template, BLASTp was performed against PDB. An automated
approach [19,20] of the MODELLER program was used to comparative
protein structure modelling by satisfaction of spatial restraints and
molecular mechanics. PROCHECK statistics and Verify 3D program were
applied to evaluate the target model. Specifically, for PROCHECK, en-
ergy rules of stereochemical were used to judge the rationality of the
protein model. The Verify 3D program mainly evaluated protein models
by calculating the compatibility of a protein’s 3D model with its own
2.3. Enzyme activity assay
For all of the substrates, β-glucosidase activity was determined ac-
cording to the method described by Dong [18] with slight modifications.
Firstly, seven kinds of substrates were prepared at a concentration of
25 mM, respectively. Next, diluted and purified BGL0224 in 20 mM
sodium phosphate buffer (pH 5.0) was incubated in 500 μL of reaction
mixture containing 25 mM substrates, at 37℃ for 30 min. Then the re-
action was terminated by adding 500 L of 1 M Na2CO3, followed by
measurement of the liberated p-nitrophenol (p-NP) at an absorbance of
μ
2

J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
amino acid sequence [21].
Gromacs embedded program and Visual Molecular Dynamics (VMD).
3. Results
2.8. Molecular docking
3.1. Investigation of substrate specificity
Semi-flexible molecular docking method was applied to explore the
catalytic mechanism of BGL0224 acting on ‘p-Nitrophenyl β-D-gluco-
pyranoside’. The 3D structure of BGL0224 obtained by homology
modeling was served as a receptor structure for molecular docking.
Minimize the energy of the receptor structure to achieve a steady state.
Then, the 3D structure of the substrate ‘p-Nitrophenyl β-D-glucopyr-
anoside’ was constructed by hydrogenation. The structure was opti-
mized using the MOPAC program to calculate the atomic charge of PM3
[22,23]. Finally, Autodock Tools 1.5.6 were used to process the struc-
ture of the ligand and the receptor, respectively. The active sites were
wrapped with docking box. The number of grid points in X, Y, Z di-
rections was set to 60 × 60 × 60, the grid spacing was 0.375 Å, the
number of docking times was 100, and the remaining parameters adopt
default values [24].
The effects of different temperatures and pH on BGL0224 catalyzing
seven kinds of substrates were shown in Fig. 1. On the whole, BGL0224
had the strongest catalytic effect on ‘p-Nitrophenyl β-D-glucopyrano-
side’, followed by ‘p-Nitrophenyl β-D-cellobioside’, ‘p-Nitrophenyl β-D-
galactopyranoside’ and ‘p-Nitrophenyl β-D-xylopyranoside’. In the seven
reactions, the optimal temperatures for catalyzing ‘p-Nitrophenyl β-D-
xylopyranoside’ and ‘p-Nitrophenyl β-D-glucuronide’ were 40℃ and
45℃ respectively, and the optimal temperatures for catalyzing the other
five substrates were 50℃. Fig. 1c and d showed that the optimal pH of
BGL0224 catalyzing seven kinds of substrates were all 5.0 and the
enzyme activity of BGL0224 was significantly reduced when the pH
value was greater than 6.0. The kinetic parameters of BGL0224 acting on
seven kinds of substrates were shown in Table 1. When catalyzing the
substrate containing ‘β-’ bonds, the values of Vmax were generally larger
except ‘p-Nitrophenyl β-D-glucuronide’ and the maximum Vmax value
was 382.81 U/mg. The Km value is usually used to measure the affinity
between enzyme and substrate. Smaller Km value indicates greater af-
finity between enzyme and substrate. BGL02024 had the smallest Km
value when catalyzing the substrate ‘p-Nitrophenyl β-D-glucopyrano-
2.9. Molecular dynamics simulation
In order to verify the stability of composite system ‘BGL0224-pNPG’,
a 50 ns molecular dynamics (MD) simulation was performed on the
structure of complex. The MD simulation was performed with ‘Gromacs
2018’ program [25], under constant temperature and constant pressure
and periodic boundary conditions. Amber99SB full-atomic force field
and TIP3P water model were used. In the process of MD simulation, all
hydrogen bonds were constrained using the LINCS [26] algorithm, and
the integration step was 2 fs. Electrostatic interactions were calculated
using the Particle-mesh Ewald (PME) method [27]. The cutoff value of
non-bonded interaction was set to 10 Å and updated every 10 steps. The
‘V-rescale’ temperature coupling method [28] and the ‘Parrinello-Rah-
man’ method [29] were used to control the simulated temperature and
pressure to 300 K and 1 bar, respectively. The steepest descent method
was applied to minimize the energy of the system to eliminate close
contacts between atoms. 100 ps NVT equilibrium simulation was per-
formed at 300 K and the MD simulation of the composite system was
performed at 50 ns. Visualization of simulation results were done using
side’, which was 0.34
μM. The ratio of Kcat to Km is an important
parameter for measuring the catalytic efficiency of enzymes and can be
used to determine the optimal substrate for enzyme. As it was shown in
Table 1, the best substrate for hydrolysis by BGL0224 was ‘p-Nitrophenyl
β-D-glucopyranoside’ and the ratio of Kcat to Km was 1034.94 Sꢀ 1
/μM.
3.2. Fluorescence spectroscopy and quenching mechanism
Took ‘p-Nitrophenyl β-D-glucopyranoside’ as an example, the effects
of different concentrations of ‘p-Nitrophenyl β-D-glucopyranoside’ on
the fluorescence intensity of BGL0224 were shown in Fig. 2a. BGL0224
had the strongest fluorescence intensity with a emission wavelength at
439 nm. When ‘p-Nitrophenyl β-D-glucopyranoside’ was added to the
Fig. 1. Effect of different temperatures and pH on BGL0224 catalyzing seven kinds of substrates. a, b: Effect of temperature on the activity of BGL0224
catalyzing seven kinds of substrates. c, d: Effect of pH on the activity of BGL0224 catalyzing seven kinds of substrates. Vertical bars show the standard deviations of
three replicates.
3

J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
Table 1
Kinetic parameters of BGL0224 catalyzing seven kinds of substrates.
Substrates
Vmax (U/mg)
Km
(μ
M)
Kcat (Sꢀ 1
)
Kcat/Km (Sꢀ 1
/μM)
p-Nitrophenyl β-D-glucopyranoside
p-Nitrophenyl β-D-galactopyranoside
p-Nitrophenyl β-D-xylopyranoside
p-Nitrophenyl β-D-cellobioside
p-Nitrophenyl β-D-glucuronide
382.81 ± 7.76
270.88 ± 6.86
256.43 ± 6.13
359.54 ± 5.55
24.17 ± 1.63
39.27 ± 1.51
39.64 ± 1.02
0.34 ± 0.04
0.95 ± 0.13
1.10 ± 0.14
0.43 ± 0.04
1.87 ± 0.58
1.42 ± 0.26
1.70 ± 0.20
351.88 ± 7.13
248.99 ± 6.30
235.71 ± 5.63
330.49 ± 5.10
22.21 ± 1.50
36.10 ± 1.39
36.43 ± 0.94
1034.94
262.09
214.28
768.58
11.88
p-Nitrophenyl
p-Nitrophenyl
α
-D-glucopyranoside
-D-galactopyranoside
25.42
α
21.43
Values represent means ± standard error (n = 3).
Fig. 2. Fluorescence quenching analysis of BGL0224. a: Fluorescence spectra of BGL0224 in the absence and presence of different concentrations of ‘p-Nitro-
phenyl β-D-glucopyranoside’ at 303 K. b: The Stern-Volmer plot of BGL0224 quenching by various concentrations of substrates at 303 K. c: Double logarithmic plot of
BGL0224 quenching by various concentrations of substrates at 303 K.
solution of BGL0224, the intrinsic fluorescence intensity of BGL0224
was decreased gradually and regularly. In addition, with the increasing
of substrate concentration, a red shift of the peak (from 439 nm to
448 nm) was observed. The fluorescence quenching process was
described and calculated with the following Stern-Volmer equation [30]
and the results were shown in Table 2 and Fig. 2b.
5.25 × 104, 4.93 × 104, 7.51 × 104, 0.93 × 104, 1.10 × 104 and
0.78 × 104 L⋅Mꢀ 1, respectively. In addition, the values of Kq were all
much greater than 2.0 × 1010 L⋅M1⋅sꢀ 1, suggesting that the fluorescence
quenching processes were predominated by the static quenching
mechanism rather than the dynamic [31]. Then, the fluorescence
quenching data were further calculated with the following equation
[30].
F0
= 1 + KSV [Q] = 1 + Kqτ0[Q]
(1)
F0 ꢀ F
F
log
= log Kb + nlog[Q]
(2)
F
where F0 and F are the fluorescence intensities of BGL0224 in the
absence and presence of substrates; [Q] are concentration of substrates;
τ0 denotes the average lifetime of the fluorophore in the absence of the
quencher (τ0 = 1 × 10ꢀ 8 s), KSV and Kq mean the Stern-Volmer
quenching constant and the quenching rate constant, respectively. The
calculation results indicated that when seven kinds of substrates were
added to the enzyme solution, the KSV of BGL0224 were 8.07 × 104,
where F0 and F are the fluorescence intensities of BGL0224 in the
absence and presence of substrates; [Q] are concentration of substrates;
Kb is the binding constant, n denotes the number of the binding sites. The
plots of log [(F0 – F)/F] versus log [Q] were presented in Fig. 2c, and the
results were shown in Table 2. The values of n were all approximately
equal to 1, which indicated that there was only one binding site on the
enzyme for each substrate. The binding constants between BGL0224 and
seven kinds of substrates were 8.09 × 104, 4.53 × 104, 3.94 × 104,
7.62 × 104, 0.73 × 104, 0.83 × 104 and 0.73 × 104 L⋅Mꢀ 1, respectively.
Table 2
Quenching and binding parameters of BGL0224-substrates complex.
Substrates
Ksv (L⋅Mꢀ 1
)
Kq (L⋅Mꢀ 1Sꢀ 1
)
Kb (L⋅Mꢀ 1
)
n
ｘ104
ｘ1012
ｘ104
3.3. Homology modeling
p-Nitrophenyl β-D-
glucopyranoside
p-Nitrophenyl β-D-
galactopyranoside
p-Nitrophenyl β-D-
xylopyranoside
p-Nitrophenyl β-D-
cellobioside
8.07
5.25
4.93
7.51
0.93
1.10
0.78
8.07
5.25
4.93
7.51
0.93
1.10
0.78
8.09
4.53
3.94
7.62
0.73
0.83
0.73
0.99
0.98
0.95
0.96
0.86
0.88
0.86
By searching the PDB database, it was found that the crystal structure
of E.coli BglA (ID: 2XHY) [32] had high sequence identity with
β-glucosidase BGL0224. The exact sequence alignment results were
shown in Fig. 3. Generally, the known crystal structure could be used as
a template for homology modeling when the sequence identity between
the crystal structure and the target protein was more than 30 %. In this
research, the identity of the two sequences reached 57 %. Therefore, the
crystal structure of 2XHY was a very good template for homology
modeling. The results of the homology modeling and the rationality
evaluation were shown in Fig. 4. The rationality evaluation of protein
model by PROCHECK program was expressed in the form of ‘Ram-
achandran plot’ which could be used to illustrate the rotation of the
p-Nitrophenyl β-D-
glucuronide
p-Nitrophenyl α-D-
glucopyranoside
p-Nitrophenyl -D-
galactopyranoside
α
Ksv is the Stern–Volmer quenching constant and Kq is quenching rate constant. Kb
is binding constant and n is the number of binding sites.
bond between
α carbon atom and the carbonyl carbon atom to the
4

J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
Fig. 3. Sequence alignment of 2XHY and BGL0224. Similar sequences are marked by boxes and identical sequences are highlighted in red.
rotation between
α
carbon atom and the nitrogen atom bond within the
(Root Mean Square Deviation) and Rg (Radius of Gyration) [34]. RMSD
means the sum of all atomic deviations between the conformation at a
certain moment and the target conformation, and it is an important basis
for measuring whether the system is stable. The RMSD results of the
main chain atoms of the composite system within 50 ns were shown in
Fig. 5a. It can be seen from the figure that the composite system basically
reached a steady state after 40 ns, and the RMSD value of the stable
system was 0.223 ± 0.016 nm. Rg can be used to describe changes of the
composite system. The greater change of Rg means the greater expan-
sion of the system. As it was shown in Fig. 5b, due to the solvation of the
compound during the simulation progress, the Rg value of the composite
system gradually increased until it stabilized at 40 ns. The Rg value of
the stable system was 2.204 ± 0.006 nm.
three-dimensional structure of protein. The ‘Ramachandran plot’ in
Fig. 4a was divided into three regions: the allowable region (red color),
the maximum allowable region (yellow color), and the prohibition re-
gion (blank). The amino acid residues in the allowable region reached
90.14 %. Only 0.20 % of the amino acid residues were in the prohibition
region, and the remaining 99.80 % were in a reasonable range, which
conformed to the energy rules of stereochemistry [33]. The compati-
bility of BGL0224’s 3D model with its own amino acid sequence was
mainly evaluated by the Verify 3D program. The Verify 3D score of the
protein model was shown in Fig. 4b. 95.99 % of the amino acid residues
scored above 0.2, which met the requirements of the evaluation pro-
gram. According to ‘Ramachandran plot’ and Verify 3D score, the 3D
structure illustrated in Fig. 4d was reasonable and could be used as a
template for subsequent research. The flexibility of amino acids in the
protein structure can be expressed by RMSF (Root Mean Square Fluc-
The number of hydrogen bonds inside the BGL0224 (Fig. 5c) and the
hydrogen bonds between the BGL0224 and the substrate (Fig. 5d) were
separately counted during the MD simulation. It can be seen from the
two figures that the number of hydrogen bonds inside the BGL0224 was
basically stable at about 360 during the MD simulation, while the
number of hydrogen bonds between the BGL0224 and the substrate
tended to be stable at 40 ns, about 4. The number of hydrogen bonds
remained stable during the MD simulation, indicating that the structure
of the composite system did not change significantly. In addition, ac-
cording to the MD simulation trajectory, the binding energy of the
composite system was calculated (Fig. 5e). The binding energy was
mainly composed of Coulomb energy and LJ energy. The contribution of
the two energies to the binding energy was basically the same. The
binding energy of the composite system fluctuated greatly in the first
20 ns, and gradually stabilized after 40 ns, with a value of
-202.00 ± 20.72 kJ/mol.
tuation). The RMSF distribution of α carbon atoms in amino acid residue
in the structure were shown in Fig. 4c. The RMSF values of most amino
acids were below 0.2, indicating that the overall flexibility of BGL0224
was relatively low.
3.4. Molecular dynamics analysis
The aforementioned results indicated that the best substrate for hy-
drolysis by BGL0224 was ‘p-Nitrophenyl β-D-glucopyranoside’. There-
fore, in this section, ‘p-Nitrophenyl β-D-glucopyranoside’ was used as the
substrate to explore the catalytic mechanism of BGL0224. The enzyme-
substrate composite system ‘BGL0224-pNPG’ was obtained by semi-
flexible molecular docking. The stability of the composite system can
be obtained by analyzing the convergence parameters during the
simulation. The convergence parameters mainly include the RMSD
5

J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
Fig. 4. Model construction and verification of BGL0224. a: ‘Ramachandran plot’ of modeled BGL0224 protein. b: Verify 3D score profile of modeled BGL0224
protein. c: The RMSF distribution of carbon of amino acid residues in the structure of BGL0224. d: 3D structure of BGL0224, where cyan represented the alpha
helices, purple represented the beta sheets, and blue lines represented the areas of greater flexibility.
α
3.5. Binding mode and catalytic mechanism
active center of BGL0224. The hydrogen atom of water molecules bound
to the carboxyl group of Glu178, and the remaining hydroxide anions
bound to glucose, breaking the covalent bond between glucose and
Glu377 to generate the final product glucose.
In order to further understand the interaction between BGL0224 and
p-NPG, the structure of composite system ‘BGL0224-pNPG’ after MD
simulation was extracted and the binding mode was analyzed in detail
(Fig. 6a). The hydrophilic amino acids of the active pocket of BGL0224
were the main part of the binding process. These hydrophilic amino
acids mainly included Gln20, His132, Glu178 and Glu377. The sugar
moiety of the substrate was combined with hydrophilic amino acids and
formed hydrogen bonds with these amino acid residues, which played an
important role in stabilizing the glycosyl structure of the substrate to
maintain a relatively stable conformation during the catalytic process.
At the same time, in the vicinity of the three amino acid residues Tyr316,
4. Discussion
This study explored the fundamental structure–function relation-
ships of a novel β-glucosidase BGL0224 from Oenococcus oeni SD-2a and
elucidated its catalytic mechanism. Based on the present work, the
catalytic effect on seven kinds of substrates, fluorescence quenching,
homology modeling, molecular dynamics analysis, binding mode and
catalytic mechanism of BGL0224 have been carried out.
Trp351 and Tyr442 with aromatic ring side chains, a strong
π-
π
inter-
BGL0224 had catalytic effects on all seven kinds of substrates, at the
same time, it had strong steroslectivity. When catalyzing seven kinds of
substrates, the effect of acid and heat on the catalytic ability of BGL0224
was similar to other reported β-glucosidases. For example, the higher
optimum catalytic temperature indicated that BGL0224 was a thermo-
stable enzyme, which was consistent with most of the reported GH1
β-glucosidases [35,36]. In addition, enzymes are typically pH sensitive
due to the need to maintain keep catalytic groups in the correct ioni-
zation state. Here, catalytic glutamate needs to function as a general acid
and high pHs could destabilize the protonated residue and impede cat-
alytic reaction. Therefore, BGL0224 showed acidic pH optima. Gener-
ally, the pH value of wine is around 3.5. The preference of β-glucosidase
BGL0224 for acidic environment had undoubtedly broadened its
action could be formed between the nitrophenyl groups of the substrate,
and the nitro group also formed hydrogen bonds with Asn317, which
further limited the conformational changes of substrate. Therefore,
hydrogen bonding and π-π interaction were important driving force for
the combination of BGL0224 and p-NPG. The reaction process of
β-glucosidase BGL0224 hydrolyzed the glycosidic bond to generate
glucose and the corresponding ligand as shown in Fig. 6b: firstly, the
substrate p-NPG bound to the catalytic active site of BGL0224. The
carbonyl oxygen atom of Glu377 attacked the C atom of the substrate,
thereby breaking the glycosidic bond. The oxygen atom of the ligand
captured the hydrogen atom of Glu178 to form a p-Nitrophenol and then
the p-Nitrophenol was removed. After that, water molecules entered the
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Enzyme and Microbial Technology 148 (2021) 109814
Fig. 5. Molecular dynamics simulation of BGL0224 catalyzing p-NPG. a: Variation of RMSD of the composite system ‘BGL0224-pNPG’ during MD simulation. b:
Variation of Rg of the composite system ‘BGL0224-pNPG’ during MD simulation. c: Variation of hydrogen bonds within BGL0224 during MD simulation. d: Variation
of hydrogen bonds between BGL0224 and p-NPG during MD simulation. e: Variation of binging energy of the composite system ‘BGL0224-pNPG’ during
MD simulation.
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J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
Fig. 6. Binding mode and catalytic mechanism of BGL0224. a: Diagram of the binding mode of BGL0224 and p-NPG. b: Catalytic mechanism of BGL0224 acting
on p-NPG. Dashed line indicates hydrogen bonds, blue curve indicates hydrophobic interaction.
application in the wine industry. In addition, β-glucosidase BGL0224
had a stronger catalytic effect on substrates containing ‘β-’ bonds, and a
According to some recent reports, both the sugar moiety and the type of
glycosidic linkage were essential to substrate recognition [37,38]. It was
also reflected in this study. Although BGL0224 had general catalytic
effects on ‘β-’ bonds, different ligands could also affect the catalytic ef-
ficiency. The low catalytic efficiency of BGL0224 for substrate
‘p-Nitrophenyl β-D-glucuronide’ proved this. Clearly, BGL0224 had a
weaker catalytic effect on substrates containing ‘α-’ bonds. Specifically,
among the seven kinds of substrates, β-glucosidase BGL0224 had the
highest affinity for substrate ‘p-Nitrophenyl β-D-glucopyranoside’ and
the lowest affinity for substrate ‘p-Nitrophenyl β-D-glucuronide’.
8

J. Zhang et al.
Enzyme and Microbial Technology 148 (2021) 109814
strong catalytic effect on substrates containing ‘β-’ bonds except when
information will be used to engineer the enzyme for enhancing its cat-
alytic efficiency and further apply it to industrial production.
the substituent was glucuronide. In contrast, it had a weak catalytic
effect on substrates containing ‘α-’ bonds, indicating the substrate
specificity of BGL0224.
5. Conclusion
The binding between the seven kinds of substrates and BGL0224
followed a static quenching mechanism, and the binding site was pre-
dicted to be one. The fluorescent properties of protein are mainly related
to whether it contains amino acid residues with fluorescent properties.
Amino acid residues with endogenous fluorescence properties mainly
include tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).
Sequence analysis of BGL0224 indicated that its amino acid sequence
contained a total of 28 Tyr residues, 27 Phe residues and 11 Trp residues,
accounting for 5.83 %, 5.63 % and 2.29 % of total number of amino acid
residues, respectively. Therefore, the quenching mechanism can be
explored by measuring the emission spectrum of BGL0224 with different
concentrations of substrates. Fluorescence quenching is the decrease in
the quantum yield of fluorescence from a fluorophore induced by a
variety of molecular interactions with quencher molecules [39,40].
Therefore, examination of small molecule induced quenching of a pro-
tein can determine the binding mechanism. In this study, the gradual
and regular decrease in the fluorescence intensity of BGL0224 indicated
that the conformational changes of the enzyme during the reaction
process. The red shift of the peak in Fig. 2a suggested that ‘p-Nitrophenyl
β-D-glucopyranoside’ quenched the intrinsic fluorescence of BGL0224
and increased the polarity of the fluorophore microenvironments [41].
These findings suggested that there were interactions and strong affinity
between BGL0224 and seven kinds of substrates, especially for the
substrate ‘p-Nitrophenyl β-D-glucopyranoside’ and ‘p-Nitrophenyl
β-D-cellobioside’, which was similar to the results of ‘substrate speci-
ficity’ section.
In conclusion, higher catalytic temperature and lower pH value were
more suitable for BGL0224. On the one hand, BGL0224 had catalytic
effects on all seven kinds of substrates, especially for substrates con-
taining ‘β-’ bonds. On the other hand, it showed somewhat substrate
selectivity. ‘p-Nitrophenyl β-D-glucopyranoside’ was considered to be
the most suitable substrate, which is similar to the result of fluorescence
quenching analysis. The homology modeling and subsequent verifica-
tion results showed that the three-dimensional structure of BGL0224
conformed to the energy rules of stereochemical. The MD simulation
results for complex ‘BGL0224-pNPG’ indicated that its overall confor-
mation stabilized at 40 ns. Two glutamic acid residues ‘Glu178 and
Glu377’ were the core of catalytic process. Overall, this study gave
specific insights on the catalytic mechanism of BGL0224, which offered
theoretical basis for its industrial application.
Funding
This work was supported by the grant of National Natural Science
Foundation of China (31871769).
CRediT authorship contribution statement
Jie Zhang: Conceptualization, Investigation, Data curation, Writing
- original draft. Ning Zhao: Investigation. Junnan Xu: Software, Data
curation. Yiman Qi: Conceptualization. Xinyuan Wei: Writing - review
& editing. Mingtao Fan: Conceptualization, Supervision, Writing - re-
view & editing.
The evaluation results showed that the 3D structure of BGL0224
constructed with the crystal structure of 2XHY as a model possessed
good stability. Fig. 4d showed the overall structure of BGL0224. There
was a (β/α)8-barrel domains that contained the active site. It was
Declaration of Competing Interest
somewhat different from the double-barrel structure of GH3 β-glucosi-
dase in some plants [42]. And the overall structure of BGL0224
exhibited low flexibility. The main reason was that the middle β-sheets
The authors report no declarations of interest.
were wrapped by more α-helices and the overall structure was relatively
stable. In addition, amino acids with greater flexibility were mainly
distributed in Gly24～Asn29, Ala42～Ala46, Lys200～Ser204, Ser250～
Ala254, Ser323～Val339, Ser347～Trp351 regions, which are located in
the ‘loop’ region of BGL0224’s 3D structure. The ‘loop’ region was
exposed to water environment and had a strong interaction with the
surrounding water molecules, so its flexibility was greater than other
regions.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.enzmictec.2021.10
9814.
References
Catalytic reaction process of BGL0224 acting on p-NPG conformed to
double displacement mechanism. There are four types of non-covalent
inter-actions that could play a key role in ligand binding to proteins:
hydrogen bonds, van der Waals forces, electrostatic forces and hydro-
phobic bonds interactions. In this study, hydrogen bonding was
considered to be the most important non-covalent inter-actions to
maintain the high-level structural stability of BGL0224. By analyzing the
convergence parameters in the simulation process, it was found that the
composite system tends to be stable after 40 ns. Compared to some other
reported complex [43,44], ‘BGL0224-pNPG’ possessed a relatively low
binding energy, indicating that the composite system ‘BGL0224-pNPG’
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mic acid residues ‘Glu178 and Glu377’ play a vital role in the whole
catalytic process. This provided a theoretical basis for the subsequent
molecular modification of the β-glucosidase. We hope that the structural
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