Purification and characterization of a glucose-tolerant β-glucosidase from black plum seed and its structural changes in ionic liquids

Article abstract of DOI:10.1016/j.foodchem.2018.09.007

The objective of this study was to characterize a plant origin β-glucosidase from black plum seeds and identify its conformational changes in twenty-six imidazolium- and amino acid-based ionic liquids (ILs). The results revealed that the purified 60 kDa e

Full text of DOI:10.1016/j.foodchem.2018.09.007

Accepted Manuscript
Purification and characterization of a glucose-tolerant β-glucosidase from black
plum seed and its structural changes in ionic liquids
Yanhong Bi, Chun Zhu, Zhaoyu Wang, Hongzhen Luo, Ruiping Fu, Xiaojuan
Zhao, Xiangjie Zhao, Ling Jiang
PII:
S0308-8146(18)31572-3
DOI:
https://doi.org/10.1016/j.foodchem.2018.09.007
FOCH 23494
Reference:
Food Chemistry
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Revised Date:
Accepted Date:
8 March 2018
27 July 2018
1 September 2018
Please cite this article as: Bi, Y., Zhu, C., Wang, Z., Luo, H., Fu, R., Zhao, X., Zhao, X., Jiang, L., Purification and
characterization of a glucose-tolerant β-glucosidase from black plum seed and its structural changes in ionic liquids,
Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.09.007
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Purification and characterization of a glucose-tolerant β-glucosidase from black
plum seed and its structural changes in ionic liquids
Yanhong Bi a, Chun Zhu a, Zhaoyu Wang b, c,*, Hongzhen Luo a, Ruiping Fu a, Xiaojuan Zhao b,
c, Xiangjie Zhao a, Ling Jiang d
a School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huai’an 223003,
P.R. China
b Faculty of Chemical Engineering, Huaiyin Institute of Technology, Huai’an 223003, P.R. China
c
Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research, Huaiyin
Institute of Technology, Huai’an 223003, P.R. China
d
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, P.R.
China
*
Corresponding authors at: Faculty of Chemical Engineering, Huaiyin Institute of Technology,
Huai’an 223003, P.R. China.
E-mail addresses: biowzy@126.com (Z.-Y. Wang)
1

Abstract
The objective of this study was to characterize a plant origin β-glucosidase from black plum
seeds and identify its conformational changes in twenty-six imidazolium- and amino acid-based
ionic liquids (ILs). The results revealed that the purified 60 kDa enzyme was monomeric in nature,
maximally active at 55 °C and pH 5.0, and nearly completely inhibited by Hg2+ and Ag+.
Attractive peculiarities of the relative low kinetic and higher glucose inhibition constants
(Km=0.58 mM [pNPG]; Ki=193.5 mM [glucose]) demonstrated its potential applications in food
industry. Circular dichroism studies showed that the secondary structural changes of the enzyme
depended not only on the anions, but also on the cations of the assayed ILs. Interestingly, no
corresponding relations were observed between the changes in enzyme structure induced by ILs
and its catalytic activities, suggesting that the influences of ILs on enzymatic processes don't rely
simply on enzyme conformational changes.
Keywords: β-Glucosidase; Enzyme structure; Ionic liquids; Kinetics
2

1. Introduction
β-Glucosidases (E.C. 3.2.1.21) capable of catalyzing the synthesis and hydrolysis of
glycosidic linkages are of pivotal importance to plants, animals, and microorganisms in producing
functional secondary metabolite glycosides (Cairns et al., 2015; Guadamuro et al., 2017). Over the
last two decades, their potential applications have become a hot research topic in food,
pharmaceutical, and bioenergy industries (Guo et al., 2015). For example, the competitive
advantages of β-glucosidase obtained from various sources, in particular, the specificity of the
enzyme, have been extendly used in liberating flavor compounds from nonvolatile or odorless
glycosides in producing wine and fruit juice, synthesizing functional oligosaccharides, and
converting glycosides into their corresponding bioactive aglycones, etc (Guadamuro et al., 2017;
Guo et al., 2015; Shin and Oh, 2014). Nowadays, β-glucosidase-mediated chemical manufacturing
is considered a promising and alternative strategy in organic reactions (Sears and Wong, 2001;
Yang et al., 2015).
Generally, β-glucosidases from various sources exhibit different reverse hydrolysis and
transglucosylation activities during the one-step synthesis of aryl- and alkyl-glycosides,
diglucosides, oligosaccharides, and glycoconjugates. However, further studies show that although
enzymatic transglucosylations usually exhibit satisfactory product yields or selectivity, the
enzyme-poor sources, expensive activated sugar precursors (i.e., UDP-α-D-glucose) (Sears and
Wong, 2001), and high cost of synthesis inevitably limit their industrial biocatalytic applications.
Most recently, β-glucosidases from abundant, inexpensive, and available plant seeds have been
found superior to other enzymes for synthesizing glycosides and their analogues by reverse
hydrolysis (Yang et al., 2015). Moreover, compared to microbial origin β-glucosidases, enzymes
3

of plant origin exhibit unique characteristics, including a high glucose tolerance, broad substrate
spectrum, and excellent selectivity (Cao et al., 2015; Yang et al., 2015). Thus far, a series of
β-D-glucopyranosides have been successfully synthesized using plant origin β-glucosidases (Lu et
al., 2010; Yang et al., 2012).
With the development of sustainable solvents for green chemistry, ionic liquids (ILs), which
possess many potential applications and advantages, are now well recognized and widely
considered as useful alternatives to classical solvents or water in biocatalysis (Hallett and Welton,
2011). Enzymatic glycosylation, transglycosylation, transgalactosylation, and saccharification
have been demonstrated by several research groups in various neat ILs or IL solution systems
(Goswami et al., 2016; Kudou et al., 2014; Xu et al., 2016). For example, Kudou et al. studied
cellobiose hydrolysis with β-glucosidase from Thermotoga maritime in a phosphate buffer
containing several ILs and the results showed that 1-butyl-3-methylimidazolium acetate
([C4MIm][OAc]) presented a positive effect on improving the enzyme activity (Kudou et al.,
2014). Our previous studies also revealed that the salidroside yield catalyzed by black plum seed
β-glucosidase could be significantly enhanced when 1-hexyl-3-methylimidazolium
tetrafluoroborate ([C6MIm][BF4]) was used as the co-solvent; further, the enzyme behavior
depend not only on the alkyl chain lengths of the cation, but also on the type of the anion in
imidazolium-based ILs (Bi et al., 2012).
Nevertheless, although ILs have attracted tremendous attention from the academia as well as
the industry, their impact on the mechanism of biocatalysis process is still not completely
understood, especially the relationship between catalysts and the physicochemical properties of
ILs. In the case of quantitative and qualitative analysis of the conformational changes induced by
4

ILs in enzyme proteins during the course of biotransformations, most of the recent work focuses
solely on lipase, α-chymotrypsin, tyrosinase, and lysozyme (Esquembre et al., 2013; Lai et al.,
2011; Liu et al., 2011; Reddy et al., 2015), while there are very few reports available on the
interactions between β-glucosidase and ILs. Encouraged by this, based on the enzyme purification,
characterization, and secondary structure analysis via circular dichroism (CD) spectroscopy, the
present study aims to provide a possible explanation for the observed excellent performance of the
black plum seed β-glucosidase in IL-containing systems for preparing β-D-glucopyranosides (Bi et
al., 2012), which might be beneficial to understand the effect of composition, structure, and
physicochemical properties of the ILs on the enzymatic glycosylation process.
2. Materials and methods
2.1. Materials
Black plum seeds were obtained from local food processing company. p-Nitrophenyl
β-D-glucopyranoside (pNPG), p-nitrophenol, choline hydroxide, [C2MIm][BF4], [C4MIm][BF4],
[C6MIm][BF4], [C2MIm][PF6], [C6MIm][PF6], [C8MIm] [PF6], [C4MIm][Cl], [C4MIm][Br], and
[C4MIm][I] were purchased from Sigma-Aldrich. All other solvents were from commercial
sources and of the highest purity available.
2.2. Extraction and purification of β-glucosidase
Crude β-glucosidase from black plum seeds was extracted according to the method of Chen
et al (Chen et al., 2013). (NH4)2SO4 was then slowly added to the crude enzyme extract to achieve
40% saturation in an ice bath, and after centrifuging (Avanti J-26XP, Beckman Coulter, USA) at
11 000 g for 15 min and removing the precipitate, more (NH4)2SO4 was added to 65% saturation
and the mixture was left to stand at 4 °C overnight. Thereafter, the mixture was centrifuged to
5

collect the protein precipitation, which was dissolved in a small volume of 20 mM Tris-HCl buffer,
pH 7.4 (buffer A) and dialyzed against the same buffer for 24 h.
The dissolved sample was submitted to a DEAE-52 anion-exchange chromatography (20
cm×1.6 cm) previously equilibrated with buffer A. Stepwise elution was used with increasing
concentrations of NaCl (0.1, 0.15, and 0.2 M) dissolved in buffer A at a flow rate of 1.5 mL/min
and the elution was monitored at 280 nm. Active fractions were pooled and applied onto a phenyl
Sepharose CL-4B hydrophobic chromatography column (20 cm×1.6 cm) equilibrated with buffer
A containing 1.0 M (NH4)2SO4 and eluted using the same buffer at a flow rate of 1.5 mL/min, with
decreasing concentrations of (NH4)2SO4 (0.75, 0.5, and 0 M). Enzyme fractions were again pooled,
concentrated using an Amicon Ultra 10-kDa cutoff centrifugal filter device (Millipore, Bedford,
MA), applied to a Sephadex G-75 (100 cm×1.6 cm) column, and eluted at a flow rate of 0.5
mL/min with the equilibrated buffer A. Active fractions were pooled and stored at 4 °C. Protein
concentration was estimated by the Bradford method with bovine serum albumin as the standard
protein.
2.3. Assay of β-glucosidase activity
The analysis procedure was carried out according to Chen et al. (Chen et al., 2013), with
slight modifications. pNPG was used as the substrate to determine the enzyme activity. An assay
reaction mixture containing 0.1 mL enzyme solution, 2.9 mL citric acid-Na2HPO4 buffer (50 mM,
pH 7.0), 15 µL pNPG (200 mM) was incubated at 45 °C for 5 min followed by adding 3.0 mL
Na2CO3 (1.0 M) to inactivate the enzyme and measuring the absorption at 405 nm. One unit of
activity (U) was defined as the amount of enzyme required to produce 1.0 μmol p-nitrophenol in
1.0 min under the above conditions.
6

2.4. Molecular mass determination
The subunit molecular mass of β-glucosidase was examined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Mini-PROTEAN Tetra Cell
(Bio-Rad, USA) with 12% polyacrylamide gels, as described by Laemmli (Laemmli, 1970). The
pre-stained protein ladder markers (Takara, Japan) with the apparent molecular weights from 10 to
100 kDa were used to evaluate protein molecular size. Bands from SDS gels were stained with
coomassie blue for visualization. The native molecular mass was measured with size exclusion
chromatography on a SephacrylS-300 HR column (GE Healthcare, Piscataway, NJ, USA) by
comparing it with the elution volume of the reference proteins. The enzyme solution was
submitted to the column and eluted with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl
at a flow rate of 0.5 mL/min. Standard molecular weight proteins used for calibration were
ribonuclease A (13.7 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), conalbumin (75
kDa), and aldolase (158 kDa) obtained from GE Healthcare.
2.5. Effect of temperature
To identify the optimum temperature for enzyme activity, the reactions were conducted at
different temperatures (25−70 °C) in citric acid-Na2HPO4 (50 mM, pH 7.0). Aliquots of the
mixture were withdrawn at specified intervals and assayed according to the method described
earlier. Thermostability was ascertained by incubating the enzyme at the given temperature (45 °C,
50 °C, 55 °C, and 60 °C, respectively) over a duration of 480 min without any substrate and
assaying the residual activities. The maximum activity of the unincubated enzyme obtained was
defined as 100%.
2.6. Effect of pH
7

The effect of pH on the purified β-glucosidase activity was investigated in 50 mM citric
acid-Na2HPO4 phosphate buffer with various pH values ranging from 3.0 to 8.0 at 55 °C. After
incubating the enzyme (0.1 mL, 0.3 mg/mL) in the buffer for 24 h, the residual enzyme activity
was measured to assess the pH stability.
2.7. Effect of metal ions and chemical reagents
The purified enzymes were preincubated in eight metal ion (Ca2+, Mg2+, Mn2+, Zn2+, Cu2+,
Hg2+, Co2+, and Ag+) solutions at 4°C for 1.0 h, and then the residual activities were examined. In
addition, five chemical reagents (EDTA, DTT, SDS, Triton X-100, and Tween 80) were added
directly to the reaction mixture containing the pNPG used as substrate to estimate their effects on
the activity. Enzyme activity in absence of metal ions or reagents was recorded as 100%.
2.8. Kinetic parameters and inhibition constants determination
Enzyme kinetic parameters were tested with pNPG as the substrate in 50 mM citric
acid-Na2HPO4 buffer pH 5.0 at 55 °C. The Km and Vmax values were determined from the
Lineweaver-Burk plots using different pNPG concentrations (0.33−8.61 mM). Besides, dixon plot
analysis was also performed to measure the glucose (48.7−496.7 mM) inhibition constant (Ki) of
the purified β-glucosidase.
2.9. Preparation of amino acid-based ILs
A group of biocompatible [choline][amino acid] ([Ch][AA]) ILs, such as [Ch][Trp],
[Ch][Asp], [Ch][Lys], [Ch][His], [Ch][Ile], [Ch][Gln], [Ch][Leu], [Ch][Phe], [Ch][Met], [Ch][Pro],
[Ch][Arg], [Ch][Glu], [Ch][Val], [Ch][Ala], [Ch][Thr], [Ch][Gly], and [Ch][Ser] were synthesized
using the method developed by Liu et al (Liu et al., 2012).
2.10. Circular dichroism spectroscopy study
8

CD spectra studies were explored based on a MOS-450/AF-CD (Bio-Logic, France) in the
far-UV region (200−250 nm), at 297 K. Each sample was placed in a 0.1 cm pathlength quartz
cuvette filled with the phosphate buffer (50 mM, pH 5.0) containing enzyme protein (0.3 mg/mL)
and different ILs (1.0%, v/v), and equilibrated at the corresponding temperature for 30 min before
analysis. The scanning rate and spectral resolution were 50 nm/min and 0.1 nm, respectively.
Sample spectra were obtained by subtracting the blank buffers (with ILs) from the experimental
spectrum and accumulated at three times. The protein second structure (like α-helix and β-sheet)
was estimated by using CDPro software package.
3. Results and discussion
3.1. Purification of β-glucosidase
A four-step procedure was explored at 4 °C to purify black plum seed β-glucosidase. After
the initial purification of the ammonium sulfate fractionation, the second DEAE-52
anion-exchange chromatography pattern exhibited three peaks corresponding to β-glucosidase
activity (see Supplementary material), namely β-glucosidase-1 (BPG for short), β-glucosidase-2,
and β-glucosidase-3, respectively. Finally, after subjecting the major fraction of active BPG to
sequential phenyl Sepharose CL-4B hydrophobic chromatography and Sephadex G-75 size
exclusion chromatography, homogeneous purification was completed. The results are summarized
in Table 1. The enzyme was purified by 64.8 fold to a specific activity of 9.1 U/mg with a yield of
1.2%.
3.2. Molecular weight determination
SDS-PAGE as well as size exclusion chromatography were carried out in order to check the
purity and molecular weight of the enzyme protein. As shown in Fig. 1A, the purified enzyme
9

appeared as a single protein band in the SDS-PAGE spectrum, suggesting homogeneous enzyme
preparation. Size exclusion chromatography on a SephacrylS-300 HR column with reference
proteins revealed that the native molecular mass of BPG was 61 kDa (Fig. 1B), which was almost
the same as the single 60 kDa-polypeptide identified by SDS-PAGE. Therefore, the native BPG
was identified as a monomeric protein; this observation is similar to previous findings on the
subunit structure of several plant β-glucosidases such as prunus domestica seed, soybean okara,
and chayote (Chen et al., 2013; Chiou et al., 2010; Mateos et al., 2015).
3.3. Effect of temperature and pH on purified BPG
The chromogenic substrate pNPG was selected as a model reactant to assess the effect of
temperature and pH on the hydrolytic activity of the purified BPG. It can be clearly seen that the
optimal temperature for enzyme activity was 55 °C (Fig. 2A), which is lower than those (60 °C
and 70 °C, respectively) of β-glucosidases from almond and apple seeds, but higher than those
(45−50 °C) from soybean okara and chayote (Chiou et al., 2010; Mateos et al., 2015; Yu et al.,
2007). Regarding the thermal stability, Fig. 2B illustrates that the catalytic activity of BPG
decreased drastically during a 480 min-incubation period at the optimal temperature, with only
50.0% of its original activity being retained after 120 min. Fortunately, a good residual activity of
about 77.2% could be achieved at 45 °C even after a the prolonged 480 min-incubation period.
Of the tested pH values ranging from 3.0 to 8.0 depicted in Fig. 2C, the acidic environment
(pH 4.0−6.0) seems to be advantageous for BPG in the production of p-nitrophenol; the optimum
pH was 5.0, which was relatively low compared to the pH required by most microbial origin
β-glucosidases (Chan et al., 2016). Moreover, the pH stability assay shows a slight reduction in the
enzymatic activity in the pH range of 5.0−8.0. In particular, after 24 h of incubation, BPG from
10

black plum seeds still maintained 90.4% residual activity, thereby indicating its excellent pH
stability and potential industrial applications.
3.4. Effect of metal cations and reagents on purified BPG
Studies on the influence of metal ions and reagents on enzyme activity were also carried out
under the reaction conditions described above. The results in Table 2 show that among the eight
metal ions examined, the catalytic activity of BPG was clearly enhanced by divalent cations like
Mn2+, Ca2+, and Cu2+ at a concentration of 5 mM, while no significant inhibition occurred in the
presence of Zn2+, Mg2+, and Co2+. However, like most β-glucosidases reported (Chen et al., 2013;
Krisch et al., 2012; Maitan-Alfenas et al., 2014; Yu et al., 2007), the sulfhydryl oxidants of Hg2+
and Ag+ nearly completely inactivated the enzyme, which suggestes that both types of ions are
competitive to the substrate pNPG. In contrast, it was interesting to find that the thiol reducing
agent DTT exerted a very slight effect on the enzyme. This means that the thiol groups in BPG
protein may not participate in the catalytic reaction, which agrees well with observations made in
previous studies (Chen et al., 2013; Lucas et al., 2000). Furthermore, the chelating agent EDTA
had no effect on the purified enzyme, implying that this purified BPG is a non-metallic enzyme.
3.5. Studies on kinetic and glucose inhibition constants
To comprehensively analyze the enzyme derived from black plum seeds, the kinetic constants
(Km and Vmax) corresponding to the activity of BPG against pNPG were measured by the
Lineweaver-Burk method. As shown in Fig. 3A, the Km and Vmax values of the enzyme were
estimated to be 0.58 mM and 90.2 μmol/(min·mg protein), respectively. Obviously, the Km value is
significantly lower than that of other well-known β-glucosidases from plant sources, such as the
apple (1.2 mM) (Yu et al., 2007), almond (2.24 mM) (Romeu et al., 1994), and prunus domestica
11

seeds (3.09 mM) (Chen et al., 2013), which indicates that BPG exhibits an extremely high
substrate specificity.
It has been well demonstrated that competitive inhibition by glucose is a common peculiarity
of β-glucosidases obtained from various sources (Cao et al., 2015; Li et al., 2012). Most microbial
β-glucosidases, usually present relatively low Ki values, which is considered a major obstacle
limiting their industrial applications. Therefore, it is particularly important to discover novel
β-glucosidases, which have a high resistance towards glucose inhibition. Dixon plots for the pNPG
hydrolysis by BPG are shown in Fig. 3B. Surprisingly, BPG afforded a much higher Ki value of
193.5 mM compared to that of most microbial β-glucosidases (0.6−8.0 mM) (Sinha and Datta,
2016). Furthermore, to the best of our knowledge, normally only plant origin β-glucosidases show
such a high tolerance to glucose. This suggests that the β-glucosidase developed in this study is
potentially useful in industrial applications.
3.6. Effect of ILs on the enzyme structure
Although biocatalysis in ILs has been the focus of extensive research and lots of work have
been reported on the interactions between enzyme proteins and ILs using different strategies
(Kumar et al., 2017), no empirical rules could be established to guide the rational choice of ILs in
biocatalytic processes. It is usually time-consuming to find an appropriate IL for a given function
by using the trial and error approach. For example, Yang et al. reported that the addition of a
certain amount of [C4MIm][I] into the reaction mixture could increase the yields of arylalkyl
β-D-glucopyranosides catalyzed by β-glucosidase by 0.2−0.5 fold (Yang et al., 2012), while Reddy
and co-workers observed the unfolding and denaturation of α-chymotrypsin protein in the same IL
(Reddy et al., 2015). Furthermore, such denaturation could be successfully offset by adding
12

[C4MIm][Br], which usually acts as a powerful devitalizer for biocatalysts. The reason for this
phenomenon remains unknown.
Therefore, nine imidazolium-based ILs were firstly selected to elucidate the molecular basis
of the impact of IL physicochemical properties on enzyme behavior. As shown in Fig. 4A and 4B,
secondary structural changes in the protein not only depend on the anions of the ILs, but also on
the cations. The α-helix contents of the protein treated with the chosen ILs were lower than that of
the protein in buffer (20.4%) (Fig. 4A), indicating that the protein unfolding and flexibility
enhancement occurred. The increased exposure of the hydrophobic cavity and perturbation of the
microstructure caused by various ILs in protein might account for the decreases of the α-helix
contents (Shu et al., 2011). Most interestingly, upon elongating the alkyl chain length of the
-
cations of PF6 -based ILs from C2 to C8, the α-helix content gradually increased from 9.2% to
11.4%, while the β-sheet content decreased (Fig. 4B). However, when the anion in the ILs
-
-
changed from PF6 to BF4 , the α-helix content decreased while that of β-sheet increased, unlike
-
the case with PF6 -based ILs. In our early study, [C6MIm][BF4] has been proven to be the best
co-solvent to achieve a high enzyme activity compared to the other ILs (Bi et al., 2012).
Obviously, the experimental data clearly shows that there was no corresponding relation between
the secondary structural changes and catalytic activity of β-glucosidase in the examined IL
systems. Similar phenomenon was also obtained with regard to the influence of twenty-four
eutectic ILs on the performance of horseradish peroxidase (Wu et al., 2014). Moreover, the
reduced of hydrogen-bond basicity of the halogen atoms (Cl > Br > I) in ILs may lead to the
observed reduction in α-helix contents.
Green [Ch][AA] ILs, which possess excellent biodegradability and low toxicity, has potential
13

applications in the fields of biotransformation and biomass pretreatment (Liu et al., 2012).
Unexpectedly, black plum seed β-glucosidase displayed a poor catalytic performance in these
novel solvents (data not shown). To further examine their impact on the catalyst, we assessed the
secondary structure of the enzyme protein in buffers containing seventeen [Ch][AA] ILs. As
shown in Fig. 4C and 4D, the protein displayed a trend similar to that of imidazolium-based ILs
with respect to the variation in α-helix and β-sheet, except in the case of [Ch][Pro]. Further, it is
worth emphasizing that these structural changes did not correlate with the properties of [Ch][AA]
ILs and the enzyme activity.
Several research groups suggested that the reduction in α-helix content and increase in
β-sheet content might affect the enzyme active region and improve its activity (Liu et al., 2011;
Wu et al., 2014). However, the results obtained in this study do not seem to comply with the above
assumptions. It is well recognized that the admirable physicochemical properties of ILs derive
from their excellent ability to form intra- and inter-molecular hydrogen bonding, which changes
the energy barrier and allows the reaction to progress (Hunt et al., 2015). Therefore, we rather
believe that not only the changes in the enzyme structure, but also the multiple interactions
occurring between the reactant, product, and solvent with ILs contribute significantly to the
biocatalysis process.
4. Conclusions
In conclusion, we have purified and characterized a novel β-glucosidase with desired features
of good thermal and pH stability, remarkable substrate affinity, and high glucose tolerance from
inexpensive and available black plum seeds. Further investigations indicated that the variation
-
trend of the enzyme secondary structure in PF6 -based ILs was completely opposite to that
14

-
observed in BF4 -based ILs. For amino acid-based ILs, their physicochemical properties also
showed ruleless impacts on enzyme structures. These findings will not only provide an efficient
plant origin β-glucosidase, but also enrich the fundamental theories of the ILs-biomacromolecule
relationship.
Acknowledgements
This research was sponsored by the National Natural Science Foundation of China
(31501421, 21676114), Qing Lan Project of Jiangsu Province, Six Talent Peaks Project of Jiangsu
Province (SWYY-011), and College Students Innovation and Entrepreneurship Training Projects
of Jiangsu Province (HGYCX2017012) for financial support.
References
Bi, Y.H., Wang, Z.Y., Mao, Y.Y., Zheng, S.Y., Zhang, H.J., & Shi, H. (2012). Ionic liquid effects
on the activity of β-glycosidase for the synthesis of salidroside in co-solvent systems.
Chinese Journal of Catalysis, 33, 1161-1165.
Cairns, J.R.K., Mahong, B., Baiya, S., & Jeon, J.S. (2015). β-Glucosidases: Multitasking,
moonlighting or simply misunderstood? Plant Science, 241, 246-259.
Cao, L.C., Wang, Z.J., Ren, G.H., Kong, W., Li, L., Xie, W., & Liu, Y.H., 2015. Engineering a
novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of
sugarcane bagasse at high glucose concentration. Biotechnology for Biofuels, 8, 202.
Chan, C.S., Sin, L.L., Chan, K.G., Shamsir, M.S., Manan, F.A., Sani, R.K., & Goh, K.M., 2016.
Characterization of a glucose-tolerant β-glucosidase from Anoxybacillus sp. DT3-1.
Biotechnology for Biofuels, 9, 174.
Chen, L., Li, N., & Zong, M.H., 2013. A glucose-tolerant β-glucosidase from Prunus domestica
15

seeds: purification and characterization. Process Biochemistry, 47, 127-132.
Chiou, T.Y., Lin, Y.H., Su, N.W., & Lee, M.H., 2010. β-Glucosidase isolated from soybean okara
shows specificity toward glucosyl isoflavones. Journal of Agricultural and Food
Chemistry, 58, 8872-8878.
Esquembre, R., Sanz, J.M., Wall, J.G., del Monte, F., Mateo, C.R., & Ferrer, M.L., 2013. Thermal
unfolding and refolding of lysozyme in deep eutectic solvents and their aqueous dilutions.
Physical Chemistry Chemical Physics, 15, 11248-11256.
Goswami, S., Gupta, N., & Datta, S., 2016. Using the β-glucosidase catalyzed reaction product
glucose to improve the ionic liquid tolerance of β-glucosidases. Biotechnology for
Biofuels, 9, 72.
Guadamuro, L., Flórez, A.B., Alegría, Á., Vázquez, L., & Mayo, B., 2017. Characterization of
four β-glucosidases acting on isoflavone-glycosides from Bifidobacterium
pseudocatenulatum IPLA 36007. Food Research International, 100, 522-528.
Guo, Y., Yan, Q.J., Yang, Y., Yang, S.Q., Liu, Y., & Jiang, Z.Q., 2015. Expression and
characterization of a novel β-glucosidase, with transglycosylation and exo-β-1,
3-glucanase activities, from Rhizomucor miehei. Food Chemistry, 175, 431-438.
Hallett, J.P., & Welton, T., 2011. Room-temperature ionic liquids: solvents for synthesis and
catalysis. 2. Chemical Reviews, 111, 3508-3576.
Hunt, P.A., Ashworth, C.R., & Matthews, R.P., 2015. Hydrogen bonding in ionic liquids.
Chemical Society Reviews, 44, 1257-1288.
Krisch, J., Bencsik, O., Papp, T., Vágvölgyi, C., & Takó, M., 2012. Characterization of a
β-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei.
16

Bioresource Technology, 114, 555-560.
Kudou, M., Kubota, Y., Nakashima, N., Okazaki, F., Nakashima, K., Ogino, C., & Kondo, A.,
2014. Improvement of enzymatic activity of β-glucosidase from Thermotoga maritima by
1-butyl-3-methylimidazolium acetate. Journal of Molecular Catalysis B: Enzymatic, 104,
17-22.
Kumar, A., Bisht, M., & Venkatesu, P., 2017. Biocompatibility of ionic liquids towards protein
stability: A comprehensive overview on the current understanding and their implications.
International Journal of Biological Macromolecules, 96, 611-651.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, 227, 680-685.
Lai, J.Q., Li, Z., Lü, Y.H., & Yang, Z., 2011. Specific ion effects of ionic liquids on enzyme
activity and stability. Green Chemistry, 13, 1860-1868.
Li, G., Jiang, Y., Fan, X.J., & Liu, Y.H., 2012. Molecular cloning and characterization of a novel
β-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and
glucose-tolerance from soil metagenomic library. Bioresource Technology, 123, 15-22.
Liu, Q.P., Hou, X.D., Li, N., & Zong, M.H., 2012. Ionic liquids from renewable biomaterials:
synthesis, characterization and application in the pretreatment of biomass. Green
Chemistry, 14, 304-307.
Liu, Y., Chen, D., Yan, Y., Peng, C., & Xu, L., 2011. Biodiesel synthesis and conformation of
lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents.
Bioresource Technology, 102, 10414-10418.
Lu, W.Y., Sun, X.W., Zhu, C., Xu, J.H., & Lin, G.Q., 2010. Expanding the application scope of
17

glycosidases using click chemistry. Tetrahedron, 66, 750-757.
Lucas, R., Robles, A., Alvarez de Cienfuegos, G., & Gálvez, A., 2000. β-Glucosidase from
Chalara paradoxa CH32: purification and properties. Journal of Agricultural and Food
Chemistry, 48, 3698-3703.
Maitan-Alfenas, G.P., de A. Lage, L.G., de Almeida, M.N., Visser, E.M., de Rezende, S.T., &
Guimarães, V.M., 2014. Hydrolysis of soybean isoflavones by Debaryomyces hansenii
UFV-1 immobilised cells and free β-glucosidase. Food Chemistry, 146, 429-436.
Mateos, S.E., Cervantes, C.A.M., Zenteno, E., Slomianny, M.C., Alpuche, J., Hernández-Cruz, P.,
Martínez-Cruz, R., Canseco, M.d.S.P., Pérez-Campos, E., & Rubio, M.S., 2015.
Purification and partial characterization of β-glucosidase in chayote (Sechium edule).
Molecules, 20, 19372-19392.
Reddy, P.M., Umapathi, R., & Venkatesu, P., 2015. A green approach to offset the perturbation
action of 1-butyl-3-methylimidazolium iodide on α-chymotrypsin. Physical Chemistry
Chemical Physics, 17, 184-190.
Romeu, A., Montero, M.A., Ruiz, J.A., De Maria, M.J., Jimenez, J.A., Rojas, A., & Arola, L.,
1994. Effects of glucose, ethanol, Hg (II) and Cu (II) on almond beta-glucosidase.
Biochemistry and Molecular Biology International, 33, 939-946.
Sears, P., & Wong, C.H., 2001. Toward automated synthesis of oligosaccharides and
glycoproteins. Science, 291, 2344-2350.
Shin, K.C., & Oh, D.K., 2014. Characterization of a novel recombinant β-glucosidase from
Sphingopyxis alaskensis that specifically hydrolyzes the outer glucose at the C-3 position
in protopanaxadiol-type ginsenosides. Journal of Biotechnology, 172, 30-37.
18

Sinha, S.K., & Datta, S., 2016. β-Glucosidase from the hyperthermophilic archaeon Thermococcus
sp. is a salt-tolerant enzyme that is stabilized by its reaction product glucose. Applied
Microbiology and Biotechnology, 100, 8399-8409.
Wu, B.P., Wen, Q., Xu, H., & Yang, Z., 2014. Insights into the impact of deep eutectic solvents on
horseradish peroxidase: Activity, stability and structure. Journal of Molecular Catalysis B:
Enzymatic, 101, 101-107.
Xu, J., Xiong, P., & He, B., 2016. Advances in improving the performance of cellulase in ionic
liquids for lignocellulose biorefinery. Bioresource Technology, 200, 961-970.
Yang, R.L., Wang, Z.Y., Bi, Y.H., Jia, J.B., Zhao, X.J., Liu, X.M., & Du, W.Y., 2015. Convenient
synthesis of alkyl and phenylalkyl β-D-glucopyranosides using facile and novel
biocatalysts of plant origin. Industrial Crops and Products, 74, 918-924.
Yang, R.L., Li, N., & Zong, M.H., 2012. Using ionic liquid cosolvents to improve enzymatic
synthesis of arylalkyl β-D-glucopyranosides. Journal of Molecular Catalysis B:
Enzymatic, 74, 24-28.
Yu, H.L., Xu, J.H., Lu, W.Y., & Lin, G.Q., 2007. Identification, purification and characterization
of β-glucosidase from apple seed as a novel catalyst for synthesis of O-glucosides.
Enzyme and Microbial Technology, 40, 354-361.
Zhou, P.F., Wang, X.P., Yang, B., Hollmann, F., & Wang, Y.H., 2017. Chemoenzymatic
epoxidation of alkenes with Candida antarctica lipase B and hydrogen peroxide in deep
eutectic solvents. RSC Advances, 7, 12518-12523.
19

5.5
5.0
4.5
4.0
3.5
B
Aldolase (158 kDa)
Conalbumin (75 kDa)
Ovalbumin (43 kDa)
61 kDa
Carbonic anhydrase (29 kDa)
Ribonuclease (13.7 kDa)
60
65
70
75
80
85
90
95
100
Elution volume (mL)
Fig. 1. SDS-PAGE analysis and molecular mass determination of BPG. (A) SDS-PAGE analysis
of purified protein samples from black plum seed (12% polyacrylamide). Lane 1, molecular
weight markers; Lane 2, crude enzyme extract; Lane 3, dissolution of (NH4)2SO4 precipitation;
Lane 4, fractions after anion-exchange chromatography; Lane 5, fractions after hydrophobic
chromatography; Lane 6, fractions after size exclusion chromatography. (B) Determination of
molecular mass of BPG by size exclusion chromatography. The reference proteins (■) are
ribonuclease A, carbonic anhydrase, ovalbumin, conalbumin, and aldolase, respectively. The
purified BPG (□) eluted at a volume corresponding to 61 kDa.
20

A
100
80
60
40
20
0
20
30
40
50
60
70
Temperature (C)
B
100
80
60
40
20
0
45 C
50 C
55 C
60 C
0
100
200
300
400
500
Time (min)
21

C
100
80
60
40
20
0
Activity
Stability
3
4
5
6
7
8
pH
Fig. 2. Effect of temperature and pH on BPG activity. (A) The activity was measured in the
temperature ranging from 25 °C to 70 °C in buffer (50 mM, pH 7.0). (B) To characterize the
thermal stability, the enzyme was incubated at the indicated temperatures for 480 min. (C) The
activity was measured in the pH values ranging from 3.0 to 8.0 at 55 °C. For pH stability, the
enzyme was incubated at the indicated pH values for 24 h.
22

0.06
0.05
0.04
0.03
0.02
A
50
40
30
20
10
-2
-1
0
1
2
3
1/[S] (mM-1)
0
2
4
6
8
Concentration of pNPG (mM)
B
30
20
Ki=193.5 mM
10
-200
-100
0
100
200
300
400
500
Concentration of glucose (mM)
Fig. 3. Determination of the kinetic and glucose inhibition constants. (A) Effects of different
pNPG concentrations on BPG activity (inset of Hanes-Woolf plot). The reactions were carried out
at pH 5.0 and 55°C by adding various amount of pNPG into 50 mM buffer. (B) Dixon plot of the
inhibition of the glucose. The reactions were carried out in the presence of fixed concentration of
pNPG [pNPG: 1.32 mM (■) and 5.19 mM (▲)] at pH 5.0 and 55°C.
23

A
20
15
10
5

0
6
6
6
4
4
4
I

Cl
Br

rol
nt
PF
PF
PF


BF

BF
BF




MIm
C4
Co
MIm MIm
C4
C4
MIm
C6
MIm
C2
MIm
MIm
MIm
C2
MIm
C8
C6
C4
B
50
40
30
20
10
0

4
6
4
4
6
6
I

Cl
rol
nt
PF
BF

Br

PF
PF
BF
BF






MIm
C4
Co
MIm
C4
MIm
MIm
C4
MIm
MIm
C6
MIm MIm
MIm
C6
C2
C4
C8
C2
C
20
15
10
5

0
r
s
Ly
Ch
e
a
p
As
Ch
s
n
y
u
Ile

Ser

Trp
Pro
Th
Gl


Ch
rol
Ch
Al
Val

Ch
Hi

Gl
Leu Ph
Gl



Met
Arg

Ch
Ch


Ch
Ch Ch





nt
Ch
Ch
Ch
Ch
Ch
Ch
Ch
Co
270

60
50
40
30
20
10
0
D

r
e
Ph
a
p
As
s
s
Ly
Ch
n
u
y
Ile
Pro

Met

Al

Val
Ch
Hi

Ser

Trp
Th
Gl

Ch
Gl
Arg Gl

Leu
Ch
rol
Ch










nt
Ch
Ch
Ch
Ch
Ch
Ch
Ch
Ch
Ch Ch
Ch
Ch
Co
Fig. 4. Secondary structure contents of BPG in the presence of 9 imidazolium- and 17 amino
acid-based ILs obtained from CD studies.
Table 1 Summary of purification of β-glucosidase from black plum seed
Total activity Total protein Specific activity Recovery Purification
Purification step
(U)
(mg)
(U/mg protein)
0.14±0.01
0.15±0.01
1.8±0.05
(%)
(fold)
Crude extract
226.2±6.5 1583.3±10.2
100.0±0.0 1.0±0.0
26.6±0.7 1.1±0.03
20.2±0.5 12.8±0.3
(NH4)2SO4 (40-65%)
DEAE-52
60.2±1.2
45.8±1.0
412.4±8.1
26.0±0.5
3.3±0.1
phenyl Sepharose CL-4B 13.5±0.3
Sephade G75 2.7±0.1
4.1±0.2
6.0±0.2
29.5±0.7
0.3±0.01
9.1±0.3
1.2±0.04 64.8±0.8
270

Table 2 Effects of metal cations and chemical reagents on BPG activity
Reagent
No addition
Zn2+
Concentration
0 mM
Relative activity (%)
100.0±0.0
98.4±0.2
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
5.0 mM
1.0% (v/v)
5.0% (v/v)
Mg2+
95.5±0.1
Mn2+
133.3±0.3
116.8±0.3
117.0±0.6
83.5±1.1
Ca2+
Cu2+
Co2+
Hg2+
3.0±0.1
Ag+
2.4±0.1
EDTA
107.3±0.8
87.7±0.7
DTT a
SDS
66.6±0.8
Triton X-100
Tween 80
a Dithiothreitol.
49.8±1.3
90.9±0.4
Highlights


A novel β-glucosidase from black plum seeds was firstly purified and characterized.
The enzyme shows lower kinetic Km value and higher glucose inhibition capability.
270



Structural changes in protein depended on the anions and cations of ionic liquids.
Amino acid-based ionic liquids exhibited ruleless impacts on the enzyme structure.
270