Cloning and functional expression of an α-galactosidase from Yersinia pestis biovar Microtus str. 91001

Article abstract of DOI:10.1271/bbb.80145

A gene encoding a glycoside hydrolase (GH) family 36 α-galactosidase was cloned from Yersinia pestis biovar Microtus str. 91001 and expressed in Escherichia coli. The purified recombinant α-galactosidase (Aga-Y) was optimally active at 37°C and pH 6.8. Th

Full text of DOI:10.1271/bbb.80145

Biosci. Biotechnol. Biochem., 72 (8), 2203–2205, 2008
Note
Cloning and Functional Expression of an ꢀ-Galactosidase
from Yersinia pestis biovar Microtus str. 91001
1
1
1
1
1
Yanan CAO, Huoqing HUANG, Kun MENG, Peilong YANG, Pengjun SHI,
1
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1;y
Yaru WANG, Huiying LUO, Zhifang ZHANG, Ningfeng WU, and Bin YAO
1
Department of Microbial Engineering, Feed Research Institute, Chinese Academy of Agricultural Sciences,
No. 12 Zhongguancun South Street, Beijing, 100081, P.R. China
2
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences,
No. 12 Zhongguancun South Street, Beijing, 100081, P.R. China
Received March 7, 2008; Accepted May 18, 2008; Online Publication, August 7, 2008
[doi:10.1271/bbb.80145]
A gene encoding a glycoside hydrolase (GH) family 36
-galactosidase was cloned from Yersinia pestis biovar
A fragment of 2.4 kb containing the ꢀ-galactosidase
gene (Genbank accession no. BK006342), aga-y, was
amplified from the genomic DNA of Y. pestis str. 91001
and sequenced according to the putative ꢀ-galactosidase
gene (Genbank accession no. AE017042). SD sequence
(AGGAG) and protein analysis showed that aga-y has
a full length of 2,127 bp, encoding 708 amino acid
residues with a calculated molecular mass of 80.37 kDa
and a calculated pI of 5.99. There was no signal peptide
at the N-terminus of the encoded protein, Aga-Y. The
identities between Aga-Y and ꢀ-galactosidases from
Escherichia coli E24377A (ABV20016), Erwinia car-
otovora subsp. Atroseptica SCRI1043 (YP 048866),
Vibrio shilonii AK1 (ZP 01867825), Aspergillus clav-
atus NRRL 1 (XP 001275168) and Penicillium sp. F63
(ABC70181) were 59, 57, 54, 28, and 25% respectively,
suggesting that Aga-Y belongs to the GH-36 family and
is more closely identical to the ꢀ-galactosidases from
bacteria than from fungi.
ꢀ
Microtus str. 91001 and expressed in Escherichia coli.
The purified recombinant ꢀ-galactosidase (Aga-Y) was
ꢀ
optimally active at 37 C and pH 6.8. The features of
temperature profile, thermoliability, kinetics, and amino
acid composition indicated that Aga-Y had properties of
a cold-adapted enzyme.
Key words: ꢀ-galactosidase; glycoside hydrolase; Yersi-
nia pestis biovar Microtus str. 91001; char-
acterization
ꢀ
-Galactosidase (ꢀ-D-galactoside galactohydrolase;
1
)
EC 3.2.1.22) is widely distributed in nature and has
been utilized in many industries, and in medical treat-
ment and scientific research. It is also involved in
cell-wall modification during tissue development.
Based on amino acid sequence similarities, ꢀ-galacto-
2
–5)
6
)
sidases have been classified into glycoside hydrolase
For heterogeneous expression, the gene aga-y was
0
(
(
GH) families 4, 27, 36, and 57 in the CAZy database
4
amplified using primers P1 (5 -GCTCCATGGTTAT-
0
)
http://www.cazy.org/fam/acc GH.html). Most ꢀ-galac-
GAATACTACCTTTG-3 , NcoI site under lined) and
0
tosidases of GH-27 come from eukaryotes and those of
GH-36 from prokaryotes. These two families contain the
majority of known ꢀ-galactosidases.
P2 (5 -GATCTCGAGGCGACCTTCTATATTTACG-
0
GCTGAAAGCT-3 , XhoI site under lined), linked
pET-22b(+) vector, and then transformed into E. coli
BL21 (DE3; Novagen, Darmstadt, Germany). The
positive clone was grown in LB medium containing
Yersinia pestis biovar Microtus str. 91001, a non-
ꢀ
plague bacterium optimally growing at 28–30 C, has
ꢀ
unique features in carbohydrate utilization: arabinose
negative, and rhamnose and melibiose positive.7) Some
enzymes of this strain studied in our laboratory showed
properties adapted to the intestinal temperature of the
animal. Hence, the aim of this study was the cloning and
functional expression of an ꢀ-galactosidase from this
strain that has similar temperature properties and
potential application in the feed industry.
100 mg/ml of ampicillin at 18 C for 12 h after
induction with 1 mM IPTG, but no enzyme activity
ꢀ
was be detected after induction at 37 C. It is similar
to some recombinant psychrophilic enzymes that
frequently are not expressed or are quickly inactivated
ꢀ
8)
at 37 C. The cells were harvested, sonicated, and
centrifuged. Two milliliters of the supernatant (crude
enzyme), was loaded onto a HiTrap Chelating column
y
To whom correspondence should be addressed. Fax: +86-10-68975127; E-mail: yaobin@caas-bio.net.cn
Abbreviations: IPTG, isopropyl-ꢁ-D-thiogalactoside; pNP, p-nitrophenyl; pNP-ꢀ-Gal, p-nitrophenyl-ꢀ-D-galactopyranoside; SDS–PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis

2204
Y. CAO et al.
T2, but lower than ꢀ-Gal II or ꢀ-Gal III from rice.4,9)
The recombinant Aga-Y was purified 111.4-fold with a
specific activity of 21.2 U/mg. The relative molecular
weight was calculated to be 80 kDa, corresponding to
the estimated value.
The purified recombinant Aga-Y was optimally
active at pH 6.8, and retained > 60% activity at
pH 6.5–7.5 (Fig. 2A). In the test of pH stability, the
enzyme had stability in a narrow range around pH 7.0
(
Fig. 2B). The optimum temperature of Aga-Y was
ꢀ
37 C (Fig. 2C), close to that of the AgaA from
Carnobacterium piscicola BA (32–37 C) but lower
Fig. 1. Expression and Purification of Recombinant Aga-Y from
E. coli.
ꢀ
10)
3)
than most GH-36 ꢀ-galactosidases from bacteria.
ꢀ
Lane M, low molecular weight markers; lane 1, extract of E. coli
clone harboring pET-22b(+) with IPTG induction; lane 2, crude
enzyme extract of E. coli clone harboring pET-22b(+)/aga-y with
IPTG induction; lane 3, Aga-Y after purification using a HiTrap
Chelating column.
After incubation at 50 C for 2, 5, and 10 min, about
63, 10, and 3% of activity was retained respectively
þ
2þ
(Fig. 2D). Metal ions Ag and Hg inactivated Aga-Y
completely. This suggests that the Cys residue is
located at the catalytic site, since these ions have been
reported to attack Cys residues at active sites in GH-36
(
Amersham Pharmacia, Uppsala, Sweden) for purifica-
3
,9)
tion with a linear imidazole gradient of 10 to 500 mM
in phosphate buffer (20 mM phosphate, 500 mM NaCl,
pH 7.4). The enzyme activity was assayed by a
ꢀ-galactosidases.
Kinetic studies were performed
ꢀ
with pNP-ꢀ-Gal as substrate at 37 C for 2 min. The
Michaelis constant (Km) and catalytic constant (kcat)
were 0.51 mM and 3,279/s. The Km for melibiose as
substrate was 1.35 mM by the GOD-POD method, and
the values for raffinose and stachyose could not be
determined by the DNS method. A hydrolyzing experi-
ment was performed, and it showed the hydrolysis
ability of melibiose (11.68%) > stachyose (11.27%) >
rafffinose (10.63%).
2
)
modified pNP-ꢀ-Gal method and loaded into SDS–
PAGE (12% w/v, pH 8.3) (Fig. 1). One unit of ꢀ-
galactosidase activity was defined as the amount of
enzyme that released 1 mmol of pNP from pNP-ꢀ-Gal
ꢀ
2)
(
Sigma, St. Louis, Mo, USA) per min at 37 C. The
ꢀ
0
-galalctosidase activity in the crude enzyme was
.19 U/ml, higher than that of ꢀ-Gal from Thermus sp.
Fig. 2. Effects of pH and Temperature on Aga-Y Activity.
A, Effect of pH on Aga-Y activity. The effect of pH on the activity of Aga-Y was tested in 0.1 M McIlvaine buffers at pH 2.2–8.0 and Glycine-
NaOH at pH 9.0–11.0. B, Effect of pH on the stability of Aga-Y. pH stability was determined by measuring the residual activity after incubation
ꢀ
in the same buffers of various pHs at a 1:10 ratio (v/v) at 37 C for 30 min. C, Effect of temperature on Aga-Y activity. The effect of temperature
ꢀ
on the activity of Aga-Y was detected in 0.1 M McIlvaine buffer, pH 6.8, from 0 to 70 C. D, Effect of temperature on the stability of Aga-Y.
ꢀ
Thermal stability was determined by measuring residual activity after pre-incubation at 50 C for 2, 5, 10, 15, 20, and 30 min. 100% relative
value was 21.1 U/mg.

An ꢀ-Galactosidase from Y. pestis
Table 1. Optimum Temperature, Kinetic Parameters and Amino
2205
References
ꢁ
Acid Composition of Aga-Y and Four Other GH-36 ꢀ-Galactosidases
1
)
Ademark, P., Larsson, M., Tjerneld, F., and Stalbrand,
H., Multiple ꢀ-galactosidases from Aspergillus niger:
purification, characterization and substrate specificities.
Enzyme Microb. Technol., 29, 441–448 (2001).
Cao, Y., Yang, P., Shi, P., Wang, Y., Luo, H., Meng, K.,
Zhang, Z., Wu, N., Yao, B., and Fan, Y., Purification and
characterization of a novel protease-resistant ꢀ-galacto-
sidase from Rhizopus sp. F78 ACCC 30795. Enzyme
Microb. Technol., 41, 835–841 (2007).
Kondoh, K., Morisaki, K., Kim, W., Park, G., Kaneko,
S., and Kobayashi, H., Cloning and expression of the
gene encoding Streptomyces coelicolor A3(2) alpha-
galactosidase belonging to family 36. Biotechnol. Lett.,
Property
Aga-Y
Agl1
40
MelA
55
Aga36A
70
Optimum
Temperature ( C)
Kinetics
3
7
ꢀ
2
)
Km (mmol)
kcat (/s)
0.51
3,279
6,429
1.40
2,123
1,500
ND
ND
ND
4.30
608
141
Km=kcat (mmol/s)
Amino acid
Composition
Cys (%)
3
)
)
1.27
7.20
2.82
4.52
9.32
1.87
0.27
9.13
1.77
2.86
9.94
1.46
0.54
6.08
2.97
4.59
11.21
1.22
0.54
6.66
2.04
2.72
12.77
0.71
Gly (%)
Met (%)
Gln (%)
Trp+Tyr+Phe (%)
Arg/Lys ratio
2
7, 641–647 (2005).
4
Li, S., Kim, W. D., Kaneko, S., Prema, P. A., Nakajima,
M., and Kobayashi, H., Expression of rice (Oryza sativa
L. var. Nipponbare) ꢀ-galactosidase genes in Esche-
richia coli and characterization. Biosci. Biotechnol.
Biochem., 71, 520–526 (2007).
ND, Not detected in the original reports.
ꢁ
Aga-Y: from Y. pestis str. 91001, BK006342; Agl1: from Penicillium sp.
F63 CGMCC 1669, ABC70181; MelA: from Lactobacillus fermentum CRL
7
22, AY612895; Aga36A: from C. stercorarium, AB089353
5
6
7
)
)
)
Jeong, D. W., Lee, J. H., Kim, K. H., and Lee, H. J., A
food-grade expression/secretion vector for Lactococcus
lactis that uses an ꢀ-galactosidase gene as a selection
marker. Food Microbiol., 23, 468–475 (2006).
Comparisons of temperature properties, kinetic, and
amino acid composition with ꢀ-galactosidases of about
0 kDa are summarized in Table 1. Aga-Y had a lower
optimum temperature, close to some known cold-
Soh, C., Ali, Z. M., and Lazan, H., Characterization of an
ꢀ
-galactosidase with potential relevance to ripening
related texture changes. Phytochemistry, 67, 242–254
(2006).
8
1
1)
adapted enzymes, weak thermostability (lost activity
ꢀ
Song, Y., Tong, Z., Wang, J., Wang, L., Guo, Z., Han,
Y., Zhang, J., Pei, D., Zhou, D., Qin, H., Pang, X., Han,
Y., Zhai, J., Li, M., Cui, B., Qi, Z., Jin, L., Dai, R., Chen,
F., Du, Z., Lin, W., Wang, J., Yu, J., Yang, H., Wang, J.,
Huang, P., and Yang, R., Complete genome sequence of
Yersinia pestis strain 91001, an isolate avirulent to
humans. DNA Res., 11, 179–197 (2004).
at 50 C for 10 min) as compared with mesophilic Aga-
ꢀ
2)
F78 (stable at 50 C for 10 min) and thermophilic
Aga36A from Clostridium stercorarium (stable at
ꢀ
12)
7
0 C for 10 min).
Aga-Y had a lower Km and a
higher kcat value, resulting in higher kcat=Km than any
other ꢀ-galalctosidase studied (Table 1). A comparison
of amino acid compositions showed that Aga-Y had
higher contents of Gly, Met, Gln, and a higher Arg/
8) Feller, G., Molecular adaptations to cold in psychro-
philic enzymes. Cell Mol. Life Sci., 60, 648–662 (2003).
9
)
Ishiguro, M., Kaneko, K., Kuno, A., Koyama, Y.,
Yoshida, S., Park, G. G., Sakakibara, Y., Kusakabe, I.,
and Kobayashi, H., Purification and characterization of
the recombinant Thermus sp. strain T2 ꢀ-galactosidase
expressed in Escherichia coli. Appl. Environ. Microbiol.,
Lys ratio and lower total contents of Trp+Tyr+Phe
1)
Table 1).1 All these characteristics were similar to
(
1
1)
the specific properties of cold-adapted enzymes. The
results suggest that Aga-Y might be a cold-adapted
enzyme.
6
7, 1601–1606 (2001).
1
0) Coombs, J., and Brenchley, J. E., Characterization of two
new glycosyl hydrolases from lactic acid bacterium
Carnobacterium piscicola strain BA. Appl. Environ.
Microbiol., 67, 5094–5099 (2001).
Acknowledgment
This work was supported by the Chinese National
High Technology Research and Development Pro-
gram (863 Program, Grants no. 2007AA100601 and
no. 2006AA02Z220). We thank Professors R. F. Yang
and X. Y. Wang of the Chinese Academy of Military
Medical Sciences for kindly providing Y. pestis
str. 91001 genome DNA.
11) Siddiqui, K. S., and Cavicchioli, R., Cold adapted
enzymes. Annu. Rev. Biochem., 75, 403–433 (2006).
1
2) Suryani, Kimura, T., Sakka, K., and Ohmiya, K.,
Cloning, sequencing, and expression of the gene
encoding the Clostridium stercorarium ꢀ-galacto-
sidase Aga36A in Escherichia coli. Biosci. Biotechnol.
Biochem., 67, 2160–2166 (2003).