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+
–