A protease-resistant α-galactosidase characterized by relatively acid pH tolerance from the Shitake Mushroom Lentinula edodes

Article abstract of DOI:10.1016/j.ijbiomac.2019.01.051

A monomeric α-galactosidase with a molecular weight of 64 kDa was purified from fresh fruiting bodies of Lentinula edodes. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-cellulose and Q-Sepharose and a final gel-filtr

Full text of DOI:10.1016/j.ijbiomac.2019.01.051

International Journal of Biological Macromolecules 128 (2019) 324–330
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: http://www.elsevier.com/locate/ijbiomac
A protease-resistant α-galactosidase characterized by relatively acid pH
tolerance from the Shitake Mushroom Lentinula edodes
a,b,
Lijing Xu a,b, Bowen Chen b, Xueran Geng a,b, Cuiping Feng a,b, Junlong Meng a,b, , Mingchang Chang
⁎
⁎
a
College of Food Science and Engineering, Shanxi Agricultural University, Taigu 030801, China
b
Collaborative Innovation Center of Advancing Quality and Efficiency of Loess Plateau Edible Fungi, Taigu 030801, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 July 2018
Received in revised form 24 December 2018
Accepted 11 January 2019
Available online 14 January 2019
A monomeric α-galactosidase with a molecular weight of 64 kDa was purified from fresh fruiting bodies of
Lentinula edodes. The purification protocol involved ion-exchange chromatography on DEAE-cellulose, CM-
cellulose and Q-Sepharose and a final gel-filtration on Superdex 75. The purified α-galactosidase (LEGI) was
identified by LC-MS/MS. It demonstrated the optimum pH of 5.0 and temperature optimum of 60 °C towards
pNPGal. It was inhibited by Cd2+, Fe3+, Pb2+, Zn2+, Al3+, Hg2+, Cr2+, Ba2+. The LEGI activity was strongly
abolished by the chemical modification N-bromosuccinimide (NBS) at 1 mM, while significantly enhanced by
the thiol-reducing agents dithiothreitol (DTT). Moreover, LEGI showed strong resistance to protease pepsin, pa-
pain, acid protease and neutral protease. LEGI demonstrated hydrolysis towards melibiose (13.27%), raffinose
(4.75%), stachyose (2.58%), locust bean gum (0.82%) and guar gum (1.29%). The Km values of LEGI for pNPGal,
stachyose, raffinose, and melibiose were found to be 1.08, 17.24, 13.80 and 8.05 mM, respectively. Results suggest
that LEGI demonstrates potential for elimination of indigestible oligosaccharides.
© 2019 Published by Elsevier B.V.
1. Introduction
mushroom L. edodes and to compare the enzymatic properties and the
hydrolyzation ability with other mushroom α-Gal.
Recently, enzyme especially glycoside hydrolases plays an important
role in various fields. As a glycoside hydrolase, α-Galactosidases (α-Gal)
(EC3.2.1.22) cleave the nonreducing α-linked galactosyl residues in
galacto-oligosaccharides as well as polymeric galactomannans [1]. It
has been found great potential for bioindustrial applications, such
as feed [2], food [3], medicine [4], and paper pulp processing [5] etc.
α-Gal has been wildly reported among plant, mammals and microor-
ganisms. Comparing amino acid similarity, the majority of fungi α-
galactosidase are categorized into GH families 27 and 36 [6].
Lentinula edodes is widely cultivated and consumed in the Orient for
its nutritional values and pharmacological effects. Variety of bioactive
compounds isolated from L. edodes (e.g. laccase, lectin, lentinan and
phytase) have shown different functions, especially the effect of
antioxidant, antitumor, antiviral and antimicrobial [7–13]. As one of
the famous edible fungi, L. edodes is a good source of functional food
or drugs due to its biological activities and no toxicity nor serious side
effects. However, α-Gal from edible fungi have been relatively less
explored compared to other microbial sources. Therefore, the objective
of this study was to purify a novel α-Gal from the fresh shitake
2. Materials and methods
2.1. Materials and chemicals
Fresh shitake mushroom was acquired from the edible fungus center
of Shanxi Agricultural University. Carboxymethyl (CM)-cellulose,
Diethylaminoethyl (DEAE)-cellulose, Quaternary amine (Q)-Sepharose
and Superdex 75HR10/30 were purchased from GE Healthcare. All
chemicals were of analytic reagent grade from China unless otherwise
noted.
2.2. Enzyme activity assay
Assay of α-Gal activity was carried out with slightly modifications
[14]. In short, 40 μL enzyme solution and 40 μL 10 mM pH 4.6 p-
Nitrophenyl-α-D-galactopyranosides (pNPGal) were incubated at
40 °C for 10 min. The reaction was terminated by adding 320 μL
0.5 mM Na2CO3. Then the released p-nitrophenol (pNP) was measured
spectrophotometrically at 405 nm. One α-Gal unit was defined as the
amount that released one micromole pNP per min.
⁎
Corresponding authors at: College of Food Science and Engineering, Shanxi
Agricultural University, Taigu 030801, China.
E-mail addresses: mengjunlongseth@hotmail.com (J. Meng), sxndcmc@163.com
(M. Chang).
All assays were repeated three times.
https://doi.org/10.1016/j.ijbiomac.2019.01.051
0141-8130/© 2019 Published by Elsevier B.V.

L. Xu et al. / International Journal of Biological Macromolecules 128 (2019) 324–330
325
Table 1
Summary of purification procedure of LEGI.
Purification step
Protein (mg)
Activity (U/mL)
Total activity (U)
Specific activity (U/mg)
Recovery (%)
Purification fold
Extracts
D3
C1
Q1
SU1
4851.37
403.20
59.52
3.72
3.78
0.54
1.57
2.05
16.82
756.28
288.15
157.08
40.10
0.16
0.71
2.64
10.77
168.25
100.0
38.10
20.77
5.30
1.00
4.44
16.53
67.29
1051.56
0.08
13.46
1.78
2.3. Isolation of LEGI
molecular weight of the active α-Gal was calculated by the standard
curve of elution volume-lgMr. The reducing SDS-PAGE was carried out
by following the method of Laemmli [15]. The gels were visualized by
Coomassie brilliant blue staining. The molecular weight of inactive
monomer was calculated by anther standard curve of relative
mobility-lgMr.
Fresh shitake mushroom was homogenized in distilled water
(4 mL/g), kept at 4 °C overnight without agitation and then centrifuged
(15 min, 10,000 g). The 1 M pH 5.2 acetate buffer was added to the clear
supernatant to 10 mM. The supernatant was loaded on a 2.5 cm × 30 cm
column of DEAE-cellulose. The active fraction D3 enriched in α-Gal activ-
ity was eluted with the 250 mM NaCl. After dialyzed against 10 mM
pH 4.6 acetate buffer, fraction D3 was subjected to a 2.5 cm × 20 cm col-
umn of CM-cellulose. The unbounded fraction CM1 containing α-Gal ac-
tivity was collected and dialyzed. The dialyzed CM1 was injected into a
1 cm × 10 cm Q-Sepharose column with 10 mM pH 5.2 acetate buffer.
A linearity gradient elution was carried on with 0–300 mM NaCl. The
unadsorbed solution Q1 with α-Gal activity was finally put on a Superdex
75 gel filtration column by an AKTA Purifier (GE Healthcare). The buffer
used for gel filtration was 10 mM NaAc-HAc + 150 mM NaCl. The first
eluted peak (SU1) with the highest specific activity was collected.
2.5. Analysis of amino acid sequence
The purified LEGI band was excised, digested then dissolved in 0.1%
formic acid for LC-MS/MS analysis. The inner amino acid sequences of
LEGI were compared with α-Gal from other sources by Mascot. Se-
quence homologues were obtained by NCBI database.
2.6. Effect of pH and temperature on LEGI
To evaluate the optimal pH of LEGI, a series of pNPGal solution in
100 mM, pH 2.0 to 8.0 Na2HPO4-citric acid buffers were tested to replace
the standard pNPGal solution at pH 4.6. The pH stability of LEGI was
assayed by incubating LEGI in the above-mentioned buffers for 60 min
at 4 °C. The residual activity of α-Gal was tested with standard method.
The temperature optima of LEGI was analyzed at temperatures varying
2.4. Estimation of molecular weight
Molecular weight estimation of LEGI was performed by the reducing
SDS-PAGE and gel filtration mentioned above. In gel filtration, the
Fig. 1. Elution curves of LEGI. (A) Anionic exchange chromatography on DEAE-cellulose column (pH 5.2), (B) Cation exchange chromatography of fraction D3 on CM-cellulose column
(pH 4.6), (C) Anion exchange chromatography of fraction C1 on Q-Sepharose (pH 5.2), (D) Gel-filtration chromatography of fraction Q1 by FPLC on a Superdex 75 column and the
standard curve of elution volume-lgMr.

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L. Xu et al. / International Journal of Biological Macromolecules 128 (2019) 324–330
2.8. Substrate specificity
To determine the hydrolyzation ability of LEGI, various synthetic
substrates (oNPGal, pNPGal, 4-nitrophenyl β-D-glucuronide and 4-
nitrophenyl α-D-glucopyranoside) and natural substrates (raffinose,
stachyose, guar gum and locust bean gum) were added to the assay so-
lution. The α-Gal activity towards synthetic substrates was estimated
by the abovementioned assay. While the amount of reducing sugar
was measured to get the α-Gal activity against the natural substrates.
Based on the substrates of melibiose, maltose, lactose and galactose, a
glucose oxidase kit (Solarbio Life Science Co., Beijing) was used to esti-
mate the hydrolyzation ability of LEGI.
2.9. Protease treatments
A series of protease with different optimal pH values including α-
chymotrypsin, acid protease, neutral protease, papain, pepsin, protein-
ase K, trypsin and subtilisin were used to assay the resistance of LEGI
to protease mentioned above, respectively. 2 mg/mL protease was
mixed with the purified LEGI (0.05 U/mL) for 1 h at 37 °C. The control
was measured without above mentioned protease.
Fig. 2. SDS-PAGE of LEGI. Lanes: Marker, molecular mass standards; LEGI, fraction SU1
from FPLC.
2.10. Enzyme kinetic
The kinetic parameters (Km and kcat) of LEGI towards pNPGal, raffi-
nose and stachyose were determined from the plot of the reciprocal of
initial velocities against corresponding reciprocal of substrate concen-
trations using Lineweaver-Burk graphs. The inhibition constants (Ki)
of LEGI with respect to galactose and melibiose were measured using
the same method.
from 20 to 90 °C in buffers. The thermal inactivation was studied by in-
cubating LEGI at diverse temperatures (20 °C, 30 °C, 40 °C, 50 °C, 60 °C,
70 °C, 80 °C and 90 °C) for 60 min. The residual activity of α-Gal was
tested with standard method.
2.7. Effects of metal ions, chemical reagents and chemical modification
reagents on LEGI activity
3. Results and discussion
Various metal ions (1.25, 2.5, 5 and 10 mM), chemical reagents (2,
20 and 200 mM), and chemical modification reagents (0, 0.2, 0.4, 0.6,
0.8 and 1.0 mM) were incubated with appropriately diluted LEGI and
10 mM pMPGal at 37 °C for 1 h, respectively. The control was measured
without above mentioned reagents.
3.1. LEGI purification and molecular weight determination
The LEGI purification protocol was shown in Table 1, resulting in the
specific activity of 168.25 U mg−1 against pNPGal with a purification
fold of 1051. The crude extract was applied on DEAE-cellulose eluting
Table 2
Comparison of the inner peptide sequence of LEGI with other mushroom α-galactosidase.
Peptide fragment
Peptide sequence
Microorganism containing similar sequence
Microorganism
Identity
Accession number
Gal1
Gal2
Gal3
Gal4
Gal5
LPLTVTVPR
Jiangella sp. DSM 45060
Nocardioides lianchengensis
Lechevalieria fradiae
100%
100%
100%
87%
SDS81292.1
SDD63066.1
SDF34224.1
SDS43309.1
WP_010982913.1
SOE77868.1
PBD01055.1
WP_095932580.1
WP_091304565.1
WP_081655742.1
WP_103353016.1
WP_043724796.1
SFR22691.1
PWK80739.1
SCF58490.1
EDY60641.2
WP_037903073.1
SED90961.1
WP_007028446.1
WP_090070319.1
WP_030477203.1
WP_005456311.1
WP_077015633.1
WP_073969731.1
WP_073865608.1
SEO80168.1
GFSAFLNGAQVASGASVDDR
MIAPNTMGAVSPR
AVGKAGIADVFNLDK
KASAATFDILDNK
Actinopolymorpha singaporensis
Streptomyces avermitilis
Streptomyces sp. OV198
Streptomyces sp. Ag82_O1-15
Streptomyces sp. Tue6028
Amycolatopsis tolypomycina
Amycolatopsis vancoresmycina
Amycolatopsis sp. CA-128772
Kutzneria sp. 744
Lentzea waywayandensis
Lechevalieria deserti
Streptomyces sp. Ncost-T10-10d
Streptomyces sviceus ATCC 29083
Streptomyces sviceus
Streptomyces sp. Ag109_O5-10
Amycolatopsis decaplanina
Lentzea flaviverrucosa
Lentzea albidocapillata
Saccharomonospora cyanea
Kribbella sp. ALI-6-A
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
Gal6
KVVLGTGETAAGR
Gal7
Gal8
GIADLFVSSGLR
LTATIGIDDK
Gal9
Gal10
VTGASATVPVEVDVAGARHVHLK
TVALFNESGSPRR
Gal11
Gal12
TWTVRPSSPGGDRVEVAGR
LSLNSGNLTLDVSRQGR
Gal13
Gal14
Gal15
ISAQFTLDEGER
SVADTLVSR
VEAASPTR
Streptomyces sp. CB02460
Streptomyces sp. CB01249
Streptomyces rubidus
Gal16
TVAPVRVTR

L. Xu et al. / International Journal of Biological Macromolecules 128 (2019) 324–330
327
filtration column and enriched in a single peak designed as SU1
(Fig. 1D). It was shown in Fig. 2 as a single band on SDS-PAGE and
deduced to be 64 kDa. Based on its elution volume of FPLC and
SDS-PAGE, it is indicated LEGI was a monomer enzyme. This was in
good agree with most of mushroom α-Gal, such as α-Gal from
Pleurotus djamor (60 kDa) Pleurotus citrinopileatus (60 kDa) [16], and
Pseudobalsamia microspore (62 kDa) [17]. Although most of microbial
α-Gal were monomeric proteins, a thermotolerant α-Gal from
Ganoderma lucidum [18] is a 249 kDa multimeric enzyme [18]. Besides,
α-Gal from P. citrinopileatus is a heterodimer with two subunits
(33 kDa, 27 kDa) [16] (Table 3).
3.2. Amino acid sequence of inner peptide
After tryptic digestion, the peptides of LEGI were elucidated by LC-MS/
MS. Among homologues sequences were gained by blast search (Table 2),
peptide LPLTVTVPR, GFSAFLNGAQVASGASVDDR, MIAPNTMGAVSPR,
VTGASATVPVEVDVAGARHVHLK, TVAPVRVTR demonstrated 100%
homology to NPCBM-associated NEW3 domain of α-galactosidase
from Jiangella sp. DSM 45060 (SDS81292.1), Nocardioides lianchengensis
(SDD63066.1), Lechevalieria fradiae (SDF34224.1), Streptomyces sp.
Ncost-T10-10d (SCF58490.1) and Streptomyces rubidus (SEO80168.1),
respectively. Peptide AVGKAGVTDVFNLDK shared 87% identical
to NPCBM-associated, NEW3 domain of alpha-galactosidase from
Actinopolymorpha singaporensis (SDS43309.1). Although the function of
this domain is still unknown. It is associated with the NPCBM family
(pfam08305), a novel putative carbohydrate binding module found at
the N-terminus of glycosyl hydrolases and typically located between the
GH27 and NPCBM domains. Therefore, it may be concluded that LEGI
belongs to GH family 27.
3.3. Effects of pH and temperature
The maximal activity of LEGI was obtained at pH 5.0 (Fig. 3), which
was identical to that of P. djamor [14], P. microspora [17], Pleurotus florida
[19], Termitomyces eurrhizus [20], Penicillium sp. F63 CGMCC [21]
and Aspergillus terreusGR [22] (Table 3). Generally, microbial α-
galactosidase possessed the optimal pH values from pH 4.5 to pH 5.5,
which has been found to be able to survive and effectively catalyze in
the gastric. LEGI showed high-stability between pH 4.0–6.0 for 1 h,
retaining more than 95% activity (Fig.3). The optimal temperature of
LEGI was determined to be 60 °C and underwent a sharp decline
when the temperature was elevated to 70 °C (Fig. 3). The optimal tem-
perature was the same with Agaricus bisporus [23], Coriolus versicolor
Fig. 3. Effects of pH and temperature on the activity and stability of LEGI. (a) optimal pH;
(b) pH stability; (c) optimal temperature; (d) thermal stability.
fraction D3 containing α-Gal activity (Fig. 1A). Subsequently, fraction
D3 was subjected to CM-cellulose. The specific activity of fraction C1
was higher than C2 (Fig. 1B). The unbounded fraction C1 was applied
to Q-Sepharose. And a linearity gradient elution was carried on with
0–300 mM NaCl. As illustrated in Fig. 1C, the activity resided in fraction
Q1. The unadsorbed solution Q1 was finally put on a Superdex 75 gel
Table 3
Comparison of biochemical characteristics and activities of α-galactosidase from Lentinus edodes and other mushrooms.
Molecular mass
(kDa) & subunit
Optimum temperature
(°C) & pH
Inhibitor
Protective agent Substrate
L. edodes
64, monomer
45, monomer
60, 5.0
60, 4.0
EDTA, NBS, meliobiose, galactos, Ag+
Hg2+, Fe3+, Cd2+ etc.
,
DTT, Cu2+
pNPG, oNPG, melibiose, raffinose
A. bisporus [23]
C. versicolor [24]
G. lucidum [18]
P. citrinopileatus [16]
P. djamor [14]
P. florida [19]
NBS, PCMB, DTT meliobiose galactose
EDTA
pNPG, stachyose, locust bean gum, guar
gum
pNPG, oNPG, melibiose, raffinose,
stachyose
pNPG, oNPG, melibiose, raffinose,
stachyose
Ag+, Hg2+, Fe3+, Cu2+
40, monomer
60, 3.0
NBS, Pb2+, Hg2+, Cu2+ Cd2+
–
249 (56), teramer
70, 6.0
galactose, xylose, Ag+, Hg2+
Cu2+
60, (33, 27),
heterodimer
60, monomer
50, 4.4
NBS, melibiose Cd2+, Cu2+ Hg2+, Al3+
Fe3+, Ag+
,
DEPC
pNPG, melibiose, raffinose
53.5, 5.0
55, 4.6–5.0
NBS, PCMB, meliobiose, galactose, K+
,
DEPC, DIC, TNBS
pNPG, melibiose, stachyose
Cd2+, Cu2+, Hg2+, Al3+, Fe3+, Ag+
99, monomer
NBS, galactose, glucose, maltose, lactose,
Ag+, Hg2+
–
pNPG, raffinose
P. microspore [17]
T. eurrhizus [20]
62, monomer
72, monomer
55, 5.0
60, 5.0
DTT, Hg2+, Cd2+, Cu2+, Fe3+
Hg2+, Cd2+, Fe3+, Cu2+, Mn2+, Al3+
EDC, DIC, DEPC
K+, Ca2+
pNPG, melibiose, raffinose, stachyose
pNPG, oNPG, melibiose, raffinose,
stachyose
pNPG, melibiose, raffinose, stachyose,
locust bean gum, guar gum
T. matsutake [25]
47, monomer
55, 4.5
NBS, Fe3+, Cu2+, Pb2+, Mn2+, Mg2+
–

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L. Xu et al. / International Journal of Biological Macromolecules 128 (2019) 324–330
Table 4
popular chelating agents, EDTA inhibits enzyme activity by chelating
Effect of different metal ions on the activity of LEGI.
metal in enzyme active site and affecting the advanced structure. More-
over, its catalytic activity was enhanced by Cu2+, it can be concluded
that LEGI was a metalloenzyme. About 92% of its activity losses were
also detected with SDS because of the enzyme's denaturation. There
was no effect on LEGI by NaCl and (NH4)2SO4.
The LEGI was affected negligibly by glucose, lactose, maltose, sodium
acetate, sucrose and xylose. Whereas it was significantly inhibited by
melibiose and the reaction end products, galactose, which suggested
they were competitive inhibitors of LEGI. This was similar to α-
galactosidase from A. bisporus [23] and T. multijuga. Ki values were
1.33 and 33.95 mM for galactose and melibiose, respectively.
Metal ion
Relative α-galactosidase activity (%)
10 mM
5 mM
2.5 mM
1.25 mM
Ag+
16.05 0.00
62.14 0.01
49.54 0.00
87.56 0.01
0.00 0.03
19.86 0.01
84.39 0.01
71.01 0.01
88.16 0.01
0.00 0.00
23.34 0.01
90.01 0.01
85.67 0.02
89.97 0.01
0.00 0.00
43.14 0.04
123.47 0.01
95.66 0.00
0.00 0.02
44.33 0.00
84.07 0.01
90.83 0.02
99.87 0.00
99.62 0.02
42.63 0.02
41.6 0.01
26.04 0.01
94.54 0.02
98.11 0.01
95.66 0.00
0.00 0.00
57.02 0.05
115.39 0.02
101.38 0.00
0.00 0.01
60.51 0.00
82.41 0.00
95.92 0.00
98.79 0.02
100.31 0.02
43.32 0.01
54.30 0.00
Al3+
Ba2+
Ca2+
Cd2+
Cr2+
Cu2+
Fe2+
Fe3+
Hg2+
K+
33.46 0.01
133.05 0.00
57.18 0.00
0.00 0.01
36.16 0.01
129.72 0.00
70.31 0.00
0.00 0.01
32.89 0.01
89.04 0.00
81.40 0.02
100.67 0.01
96.51 0.01
36.26 0.00
35.31 0.01
33.71 0.00
85.28 0.01
89.80 0.02
100.21 0.01
97.77 0.03
37.23 0.01
38.61 0.01
3.5. Effects of chemical modification reagents on LEGI
Mg2+
Mn2+
Na+
The association of amino acid in the active site and catalytic mecha-
nism of LEGI was investigated by the modification of amino acid by
chemical reagents (Fig. 4) The LEGI activity was strongly abolished
by N-bromosuccinimide (NBS) at 1 mM in the acidic condition, which
indicated that tryptophan may participate in substrate binding. The
catalytic activity of LEGI was dramatically increased by dithiothreitol
(DTT) which indicating that the catalytic activity is thiol group
dependent. This was similar to the observation on the alkaline α-
galactosidase from Oryza sativa L. cv. Tainong 67 [27]. The single histidyl
residue, arginine residue and carboxyl groups can be modified by 2,3-
butanedione (DIC), diethylpyrocarbonate (DEPC) and carbodiimide
(EDC), respectively. Incubation LEGI with the above reagents merely
resulted in slight changes in enzyme activity, suggesting that active
center of LEGI contained no histidyl residue, arginine residue nor
carboxyl groups.
Pb2+
Zn2+
α-Galactosidase activity in the absence of metal ions was regarded as 100%. Results repre-
sent mean standard deviation (n = 3).
[24] and T. eurrhizus [20]. It was higher than that of α-galactosidase
from P. djamor [14], P. microspore [17], Tricholoma matsutake [25] and
P. florida [19], but lower than that of α-galactosidase from G. lucidum
[18] (Table 3). The LEGI was stable at 40 °C for 1 h without loss of activ-
ity. When the temperature is up to 70 °C, most of the α-Gal was
inactivated.
3.4. Effect of metal ions and chemical reagents on LEGI
With the increase of the metallic ions' concentration, the activity of
3.6. Resistance to proteases
LEGI were gradually inhibited by Ag+, Al3+, Ba2+, Cd2+, Cr2+, Fe2+
,
Fe3+, Hg2+, Pb2+, and Zn2+(Table 4). In general, most of the α-
galactosidase can be inactivated by metal ions especially Hg2+ and
Ag+ suggesting that thiol groups and carboxyl group are required for
the adequate function of LEGI. Among those metallic ions, Cd2+ and
Fe3+ were the strongest inhibitor towards LEGI, because the enzyme ac-
tivity was completely lost by at 1.25 mM. LEGI was depressed by Cd2+
As is shown in Table 6, LEGI showed strong resistance to protease
pepsin (102.80% residual activity), papain (95.65% residual activity),
acid protease (88.25% residual activity) and neutral protease (75.07%
residual activity) at 5 mg/mL. It suggested that LEGI demonstrated the
excellence resistant to proteases with optimum pH range from neutral
to acid. Since the acidic environment in animal gut, LEGI exhibited its
application potentials in the industries of food and animal feed. The
enzyme activity decreased drastically by trypsin and protease K at
concentration of 0.5 to 5 mg/mL. LEGI were hydrolyzed efficiently by
α-chymotrypsin and subtilisin at a lower concertation. This indicated
that LEGI was sensitive to trypsin, protease K, α-chymotrypsin and
subtilisin.
and Fe3+, which was similar to that purified from A. bisporus (Fe3+
)
[23], P. djamor (Fe3+) [14], T. matsutake (Fe3+) [25] and P. microspore
(Cd2+) [17]. The influence of Ca2+, K+, Mg2+, Mn2+ and Na+ on LEGI
was negligible. LEGI was found to be significantly stimulated by Cu2+
.
Various studies have been reported the inhibition of α-galactosidase ac-
tivity by Cu2+ except α-galactosidase purified from G. lucidum [18]
which was slightly stimulated [26].
The effect of chemical reagents on LEGI was displayed in Table 5. The
activity of LEGI was significantly inhibited by EDTA. As one of the most
RmGal36 displayed better antiprotease activity of trypsin, protein-
ase K and subtilisin [28]. The α-galactosidase from Gibberella sp. F57
was observed strong resistance to α-chymotrypsin, trypsin, collagenase
Table 5
Effect of chemical reagent on activity of LEGI.
Chemical reagent
Relative α-galactosidase activity (%)
200 mM
20 mM
2 mM
EDTA
SDS
NaCl
78.03 0.02
7.89 0.00
82.18 0.01
15.15 0.00
99.50 0.00
99.37 0.04
108.87 0.02
100.54 0.01
76.24 0.02
108.69 0.02
57.62 0.01
94.16 0.02
97.13 0.05
102.57 0.00
99.38 0.03
23.22 0.00
100.27 0.00
99.12 0.01
100.87 0.03
103.17 0.02
87.41 0.00
112.25 0.01
78.82 0.03
99.01 0.01
99.45 0.02
104.38 0.00
96.84 0.00
99.96 0.03
118.38 0.00
92.65 0.03
45.20 0.01
106.50 0.02
30.31 0.02
91.32 0.01
84.90 0.01
96.55 0.00
(NH4)2SO4
Sucrose
Lactose
Meliobiose
Glucose
Galactose
Sodium acetate
Xylose
Maltose
α-Galactosidase activity in the absence of chemical reagents was regarded as 100%. Results
represent mean standard deviation (n = 3).
Fig. 4. Effect of chemical reagents on the activity of LEGI.

L. Xu et al. / International Journal of Biological Macromolecules 128 (2019) 324–330
329
Table 6
Table 8
The effect of protease on LEGI.
Kinetic parameter for LEGI.
Protease
Relative α-galactosidase activity (%)
Substrate
Km (mM)
kcat (s−1
)
kcat/Km (mM−1·s−1
)
5 mg/mL
2.5 mg/mL
0.5 mg/mL
pNPGal
1.08
14.57
21.29
43.93
33.81
13.49
1.23
3.18
4.20
Stachyose
Raffinose
Melibiose
17.24
13.80
8.05
Pepsin (pH 3.0)
Papain (pH 6.0)
115.71 0.02
95.65 0.00
24.92 0.00
88.25 0.03
39.32 0.00
32.48 0.03
75.07 0.01
57.05 0.02
108.95 0.01
98.57 0.00
31.72 0.00
95.72 0.04
67.27 0.02
35.16 0.01
83.76 0.02
62.97 0.02
102.80 0.03
101.98 0.00
46.06 0.00
97.52 0.01
100.98 0.03
37.54 0.02
91.75 0.00
80.15 0.01
Subtilisin (pH 7.5)
Acid protease (pH 4.0)
Trypsin (pH 7.0)
α-Chymotrypsin (pH 7.0)
Neutral protease (pH 7.0)
Protease K (pH 7.5)
Among the natural substrates, LEGI exhibited the highest value of
the kcat/Km ratio for pNPGal followed by melibiose, raffinose and
stachyose. The higher kcat/Km ratio indicated higher hydrolytic effi-
ciency of pNPGal than RFOs.
α-Galactosidase activity in the absence of protease was regarded as 100%. Results repre-
sent mean standard deviation (n = 3).
4. Conclusion
and subtilisin [29]. It seems that different sources of α-galactosidase had
difference proteases resistance.
Lentinula edodes is rich with α-galactosidase, which was firstly
purified by various chromatography. LEGI is a 64 kDa monomeric
metalloenzyme. Considering the enzymatic properties of LEGI such as
degradation of RFOs, strong resistance to protease and good acid-
tolerant, LEGI possesses potential applications on degradation of
antinutritional factors in food and feed industries. However, the yield
of LEGI purification is low, which will limit its applications in industries.
Therefore, efforts must be made to increase the enzyme recovery. For
example, using the aqueous two-phase extraction to make the purifica-
tion process simple and efficient. Based on the reported genome
sequence of shiitake mushroom, we plan to clone and express its α-
galactosidase gene in heterologous hosts for further study.
3.7. Substrate specificity
As shown in Table 7, LEGI hydrolyzed pNPG (100%) more efficiently
than other synthetic nitrophenyl derivatives such as oNPG (9.2%), 4-
nitrophenyl α-D-glucopyranoside (8.1%) and 4-nitrophenyl β-D-
glucuronide (4.97%). It indicated that anomeric carbon of the
substrate is better access to the active site of LEGI. Compare to
synthetic substrates, raffinose family oligosaccharides such as
melibiose (13.27%), raffinose (4.75%), stachyose (2.58%) and branched
polysaccharides such as locust bean gum (0.82%) and guar gum
(1.29%) were partially hydrolyzed. This suggested that the relative
activity of LEGI towards composite substrates was higher than
that to natural substrates, consistent with α-galactosidase from
P. djamor [14], Aspergillus terreus [30]. In general, most purified α-
galactosidase did not act on the polysaccharides [17,20,24,28].
While LEGI demonstrated meager hydrolytic activity to guar gum
and locust bean gum.
Acknowledgements
This work was financially supported by Key Project of Shanxi Key
R&D Program of China (201603D21106), Shanxi Key Scientific and
Technology of Coal Basic Research Project (FT2014-03-01) and Doctoral
Science Foundation of Shanxi Agricultural University (2014YJ09,
2015YJ04). Special thanks to Uma Maheswari Rajagopalan from
Shibaura Institute of Technology for her contribution in improving the
manuscript.
3.8. Kinetic parameters of LEGI
The kinetic constants were determined through Michaelis-Menten
equation towards pNPGal, melibiose, stachyose and raffinose
(Table 8). The Michaelis-Menten constants (Km) of LEGI on pNPGal
were lowest, which indicated that the optimum substrate of LEGI was
pNPGal rather than natural oligosaccharides. The Km value of LEGI
was similar to α-galactosidase from T. matsutake [25] (0.99 mM) and
P. florida [19] (1.1 mM), lower than that of Ruminococcus gnavus E1
[31] (Km = 1.8 mM) and higher than that of most mushroom, such as
C. versicolor [24] (0.01 mM), G. lucidum [18] (0.4 mM) and P. djamor
[14] (0.76 mM) etc.
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