Molecular cloning of the gene encoding a novel beta-N-acetylhexosaminidase from a marine bacterium, Alteromonas sp. strain O-7, and characterization of the cloned enzyme.

Article abstract of DOI:10.1271/bbb.66.471

We have reported that the chitinolytic system of Alteromonas sp. strain O-7 consists of chitinases (ChiA, ChiB, and ChiC), a chitinase-like enzyme (ChiD), beta-N-acetylglucosaminidases (GlcNAcasesA, GlcNAcaseB, and GlcNAcaseC), and a novel transglycosylative enzyme (Hex99). The gene encoding a beta-hexosaminidase with an unusual substrate specificity (hex86), located upstream of the hex99 gene, was cloned and sequenced. The gene encoded a protein of 761 amino acids with a calculated molecular mass of 86,758 Da. The deduced amino acid sequence of Hex86 showed sequence similarity with beta-hexosaminidases belonging to family 20. The hex86 gene was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme rapidly cleaved p-nitrophenyl-beta-N-acetyl-D-glucosaminide and slowly cleaved p-nitrophenyl-beta-N-acetyl-D-galactosaminide. Unexpectedly, the enzyme did not hydrolyzed chitin oligosaccharides under the assay conditions for synthetic glycosides. However, after prolonged incubation with excessive quantities of the enzyme, Hex86 hydrolyzed chitin oligosaccharides. These results indicate that Hex86 is a novel enzyme that prefers p-nitrophenyl-beta-N-acetyl-D-glucosaminide to chitin oligosaccharides as a substrate.

Full text of DOI:10.1271/bbb.66.471

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Molecular Cloning of the Gene Encoding a Novel β-
N-Acetylhexosaminidase from a Marine Bacterium,
Alteromonas sp. Strain O-7, and Characterization of
the Cloned Enzyme
a
a
a
a
Hiroshi TSUJIBO , Katsushiro MIYAMOTO , Makiko YOSHIMURA , Miwa TAKATA , Junko
a
a
MIYAMOTO & Yoshihiko INAMORI
a
Osaka University of Pharmaceutical Sciences 4-20-1 Nasahara, Takatsuki, Osaka
69-1094, Japan
5
Published online: 22 May 2014.
To cite this article: Hiroshi TSUJIBO, Katsushiro MIYAMOTO, Makiko YOSHIMURA, Miwa TAKATA, Junko MIYAMOTO &
Yoshihiko INAMORI (2002) Molecular Cloning of the Gene Encoding a Novel β-N-Acetylhexosaminidase from a Marine
Bacterium, Alteromonas sp. Strain O-7, and Characterization of the Cloned Enzyme, Bioscience, Biotechnology, and
Biochemistry, 66:2, 471-475, DOI: 10.1271/bbb.66.471
To link to this article: http://dx.doi.org/10.1271/bbb.66.471
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Biosci. Biotechnol. Biochem., 66 (2), 471–475, 2002
Note
Molecular Cloning of the Gene Encoding a Novel b-N-Acetylhexosaminidase
from a Marine Bacterium, Alteromonas sp. Strain O-7,
and Characterization of the Cloned Enzyme
Hiroshi TSUJIBO,† Katsushiro MIYAMOTO, Makiko YOSHIMURA, Miwa TAKATA
Junko MIYAMOTO, and Yoshihiko INAMORI
,
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
Received September 10, 2001; Accepted October 4, 2001
We have reported that the chitinolytic system of
Alteromonas sp. strain O-7 consists of chitinases (ChiA,
cleave the insoluble chitin to give chitobiose
[(GlcNAc) ] as a major degradation end product. The
resulting (GlcNAc) is hydrolyzed by -acetyl-
2
ChiB, and ChiC), a chitinase-like enzyme (ChiD),
b
-
N
-
2
b-N
acetylglucosaminidases (GlcNAcasesA, GlcNAcaseB,
and GlcNAcaseC), and a novel transglycosylative en-
glucosaminidases at the non-reducing end.
Alteromonas sp. strain O-7 is a Gram-negative,
zyme (Hex99). The gene encoding a
b
-hexosaminidase
‰agellated, motile, and aerobic rod-shaped bacteri-
2
)
with an unusual substrate speciˆcity (hex86), located
upstream of the hex99 gene, was cloned and sequenced.
The gene encoded a protein of 761 amino acids with a
calculated molecular mass of 86,758 Da. The deduced
amino acid sequence of Hex86 showed sequence
um of marine origin. We have been studying the chi-
tin degradation system of the strain as a model for
deˆning the various components involved in chitin
use. Chitinolytic enzymes of the strain consists of
three chitinases (ChiA, ChiB, and ChiC) and
similarity with
0. The hex86 gene was expressed in Escherichia
coli, and the recombinant enzyme was puriˆed to homo-
geneity. The enzyme rapidly cleaved -nitrophenyl-
-acetyl- -glucosaminide and slowly cleaved -nitro-
phenyl- -acetyl- -galactosaminide. Unexpectedly,
b
-hexosaminidases belonging to family
three
b
-
N
-acetylglucosaminidases
(GlcNAcaseA,
3
–7)
2
GlcNAcaseB, and GlcNAcaseC). We have clariˆed
that these enzymes and the corresponding genes are
essential for the chitinolytic system of the strain. In
addition, we have recently cloned and characterized a
novel chitinase-like enzyme (ChiD) with high activity
only towards chitooligosaccharides from trimer to
hexamer (unpublished data). In chitinase-producing
microorganisms, chitin is a common inducer of
chitinase production. However, since chitin is insolu-
ble and impermeable to microorganisms, a soluble
p
b
-
N
D
p
b
-
N
D
the enzyme did not hydrolyzed chitin oligosaccharides
under the assay conditions for synthetic glycosides.
However, after prolonged incubation with excessive
quantities of the enzyme, Hex86 hydrolyzed chitin
oligosaccharides. These results indicate that Hex86 is a
novel enzyme that prefers
glucosaminide to chitin oligosaccharides as a sub-
strate.
p
-nitrophenyl-
b
-
N
-acetyl-
D
-
degradation product (s) such as GlcNAc, (GlcNAc)
or higher oligomers is considered to act as a direct
inducer of chitinase. In Altermonas sp. strain O-7,
6)-(GlcNAc) is one of the smallest molecules to
2
,
b
-
(
1
ª
2
8
)
Key words:
b
-
N
-acetylhexosaminidase;
chitin
induce chitinase production. We have analyzed the
gene encoding the enzyme (Hex99) which synthesizes
oligosaccharide; Alteromonas sp.
8
)
b
-(1
ª
6)-(GlcNAc)
2
from
b
-(1
ª
4)-(GlcNAc)
2
.
Re-
Chitin is an insoluble polysaccharide consisting of
-(1,4)-linked -acetylglucosamine (GlcNAc) units
cently, we found that a gene (hex86), located up-
stream of the hex99 gene, encoded a hexosaminidase
with an unusual substrate speciˆcity. In this study,
we describe the cloning, expression, and the charac-
terization of the recombinant enzyme.
b
N
and is the second most abundant polymer after cellu-
lose. Chitin is a major component of crustacean and
insect exoskeletons and fungal cell walls. This poly-
saccharide is a particularly important nutrient source
to maintain the ecosystem in the marine environ-
Plasmid pTG3 contains a 4.8-kb PstI-EcoRI frag-
ment of the Alteromonas sp. strain O-7 chromosome
carrying the hex99 gene encoding a transglycosylat-
1
)
ment. Chitinolytic marine bacteria play an im-
portant role in this chitin recycling process. To
degrade chitin, chitinolytic microorganisms produce
two classes of enzymes: chitinase (EC 3.2.1.14) and
8
)
ing enzyme. Sequencing analysis of the 0.35-kb
region immediately upstream of hex99 revealed a
partial open reading frame (ORF) encoding a protein
similar to the C-terminal part of hexosaminidases
b-N-acetylglucosaminidase (EC 3.2.1.30). Chitinases
†
To whom correspondence should be addressed. TEL&FAX: +81-726-90-1057; E-mail: tsujibo
＠
gly.oups.ac.jp

4
72
H. TSUJIBO et al.
Fig. 1. Restriction Map of pTG3.2.
The hybridization probe is represented by the box. The arrows indicate ORFs and directions of transcription.
9
–11)
belonging to family 20 of glycosyl hydrolases.
Therefore, cloning of the 5 upstream region of the
of Hex86 with the BLAST database showed that the
gene encoded a protein homologous to the several
hexosaminidases belonging to family 20. Hex86
showed similarity with -acetylglucosaminidase
?
b-
gene, termed hex86, was done by colony hybridiza-
tion using the 1.2-kb PstI-EcoRI fragment as a probe
Fig. 1). Southern hybridization against total DNA
b-N
1
2)
(
(52
z
identity) from Pseudoalteromonas sp. S9,
b-
digested with various restriction enzymes showed
that the probe strongly hybridized with a 3.6-kb SacI-
EcoRI fragment (data not shown). Colony hybridiza-
tion and Southern hybridization were done as in the
N
-acetylhexosaminidase (40
z
identity) from Por-
1
3)
phyromonas gingivalis
,
putative sugar hydrolase
(40
z
identity) from Streptomyes coelicolor (Gen-
Bank accession no. CAB72189),
(37 identity) from Xylella fastidiosa (GenBank ac-
cession no. B82755), and -acetylglucosaminidase
(35 identity) from Caulobacter crescentus (Gen-
Bank accession no. AAK22434). Tews et al. reported
that an -acetylglucosaminidase from Serratia mar-
b-hexosaminidase
8
)
previous paper. Chromosomal DNA of the strain
z
was digested with SacI and EcoRI and elec-
b-N
trophoresed on a 0.6
z
agarose gel. The fragments
z
corresponding to the size of 3.6 kb were excised from
the gel and puriˆed with a Sephaglas BandPrep kit
N
(
Amersham Pharmacia Biotech). These were ligated
cescens uses an acid-base reaction mechanism with
glutamic acid 540 as the catalytic amino acid, which
is well conserved in all members of family 20 glycosyl
into the corresponding sites of pUC19 and the recom-
binant plasmids were introduced into E. coli JM109.
The library was screened by colony hybridization
with the labelled probe. Among about 850 transfor-
mants, only one clone (pTG3.1) was isolated (Fig. 1).
Analyses by restriction enzyme digestion and se-
quencing of the fragment showed that the insert of
pTG3 and that of pTG3.1 shared a 1.2-kb PstI-
EcoRI region. The 2.4-kb SacI-PstI fragment of
pTG3.1 and 4.8-kb PstI-EcoRI fragment of pTG3
were ligated together. The resulting 7.2-kb fragment
was inserted into the corresponding sites of pUC19.
The plasmid was named pTG3.2.
1
4)
hydrolases. Hex86 also contains the glutamic acid
residue (Glu 339) proposed to be the proton donor
essential for catalytic activity (Fig. 3). These results
indicate that Hex86 is a member of family 20 of the
glycosyl hydrolases.
To investigate the enzymatic properties of Hex86,
the hex86 gene was overexpressed by using a plasmid,
pET-20b(+) (Novagen). Primers for ampliˆcation of
the gene, (5
CTTCAG-3 , 5
GATATACC-3
?
?
-GTTCACACCACTTTGTACAAAG-
-GTATTAGGGCTCGAGTGATT-
) were designed to facilitate cloning
?
?
The entire nucleotide sequence of pTG3.2 indicat-
ed that hex86 was transcribed from the same strand
as hex99 and the two genes were separated by 142
nucleotides (Fig. 2). The ORF has an ATG start
codon which is preceded by a plausible ribosome-
binding site (AGGAAG) at a distance of seven
nucleotides. The hex86 gene consists of 2,286 nucleo-
tides encoding a protein of 761 amino acids with a
calculated molecular mass of 86,758 Da. There is no
apparent N-terminal secretory signal sequence, which
is composed of a positively charged amino terminus
followed by a hydrophobic core and a string of polar
residues, downstream from the start site, suggesting
that Hex86 is an intracellular enzyme.
in frame into pET-20b(+). The hex86 gene was am-
pliˆed by the polymerase chain reaction (PCR) with
these primers with pTG3.2 as the template. PCR am-
pliˆcation was done out for 30 cycles (30 s at 94
9C ,
30 s at 59 C, 2.5 min at 68 C) with KOD-Plus DNA
9
9
polymerase (Toyobo, Osaka, Japan). The ampliˆed
DNA was digested by HindIII and XhoI, and the
resulting fragment was inserted into the correspond-
ing sites of pET-20b(+). This plasmid was designat-
ed pET-HEX86.
E. coli BL21 (DE3) cells carrying pET-HEX86
were induced with 1 m
M
isopropyl-
D
b- -thiogalac-
topyranoside (IPTG) at the mid-exponential phase
and further incubated for 2 h at 37 C. Cells were har-
vested by centrifugation and resuspended in phos-
9
Comparison of the deduced amino acid sequence

b
-
N
-Acetylhexosaminidase from Alteromonas sp. Strain O-7
473
Fig. 2. Nucleotide Sequence of hex86
.
The deduced amino acid sequence of Hex86 is shown below the nucleotide sequence. The putative ribosome-binding site is underlined.
The stop codon is indicated by an asterisk. The nucleotide sequence data will appear in the DDBJ, EMBL, and GenBank nucleotide se-
quence databases with the accession number AB067646.
Fig. 3. Alignment of the Active Site of Hex86 and Other
Numbers on the left are the residue numbers of the ˆrst amino acid in each line. Residues that are identical are indicated by bold let-
ters. The putative active-site glutamic acid rsidues are marked by an asterisk. Hex86, -hexosaminidase from Alteromonas sp. strain
O-7; ChiQ, -acetylglucosaminidase from Pseudoalteromonas sp. S9; NahA, -acetylglucosaminidase from Porphyromonas gin-
givalis; Pshy, putative sugar hydrolase from Streptomyces coelicolor; Hex, -hexosaminidase from Xylella fastidiosa; Nah, -acetyl-
glucosaminidase from Caulobacter crescentus
b-Hexosaminidases.
b
b
-
N
b-N
b
b-N
.
phate-buŠered saline (PBS). The cells were disrupted
by sonication, and the lysate was centrifuged at
binant Hex86 calculated from the deduced amino
acid sequence was in reasonable agreement with the
86 kDa estimated by SDS-PAGE (Fig. 4).
1
0,000
Hex86 was solubilized with PBS containing 8
The solution was centrifuged , and dialyzed against a
series of 50 m Tris-HCl buŠers (pH 7.0) with
stepwise decreases in the urea concentration from 6
to 0 . The soluble protein was put on a chelating
×
g
for 20 min. The pellet containing insoluble
M
urea.
Enzyme activity was measured using
nitrophenyl- -acetyl- -glucosaminide (PNP-
GlcNAc) in 50 m
tion mixture, composed of 2.5 m
50 m acetate buŠer (pH 5.5), and enzyme solution
in 0.15 ml of reaction volume, was incubated at 37
for 10 min. The reaction was stopped by adding
0.5 ml of 0.2 Na CO and the -nitrophenol liber-
ated was measured at 420 nm. One unit of
acetylhexosaminidase was deˆned as the amount of
p
-
b
-
N
D
b
-
M
M
acetate buŠer, pH 5.5. The reac-
M
PNP-b-GlcNAc,
M
M
Sepharose HP column (Amersham Pharmacia
Biotech) according to the manufacturer's instruc-
tions. The active fraction was further puriˆed by
Mono Q column chromatography (Amersham Phar-
macia Biotech). The molecular mass of the recom-
9C
M
2
3
p
b-N-

4
74
H. TSUJIBO et al.
Table. Hydrolysis Rate of Chitin Oligosaccharides by the Puri-
ˆed Hex86
Substrate
Di- -acetylchitobiose
Relative rate (z a
)
N
100.0
74.8
46.6
41.1
27.6
Tri- -acetylchitotriose
Tetra- -acetylchitotetraose
Penta-
Hexa-
N
N
N
-acetylchitopentaose
N
-acetylchitohexaose
a)
Rate relative to activity with
was taken as 100
N-acetylchitobiose as a substrate, which
z
.
increases in the degree of polymerization (Table). On
the other hand, ExoII and NagZ had no activity
against chitin oligosaccharides despite prolonged
Fig. 4. SDS-PAGE of Hex86.
Lanes: 1, marker proteins; 2, Hex86.
incubation with excessive quantities of these en-
16,17)
zymes.
Hex86, belonging to family 20, showed no
sequence similarity with ExoII and NagZ, belonging
to family 3. The diŠerence in amino acid sequences
of these enzymes might re‰ect the diŠerence of the
reactivity towards chitin oligosaccharides. There are
many reports on hexosaminidases from a wide varie-
ty of organisms, however, there is no report of a
family 20 hexosaminidase with the unusual speciˆcity
of Hex86.
enzyme that liberated 1
min under the conditions described above. The
optimun pH and temperature of Hex86 were 5.5 and
C, respectively. We investigated the substrate
speciˆcity of the enzyme by using various substrates.
Among synthetic glycosides, the enzyme was most
mmol of p-nitrophenol in
1
379
active towards PNP-
showed a low activity towards PNP-
galactosaminide (7.6 of the reactivity towards
PNP- -GlcNAc). However, the enzyme did not
hydrolyze PNP- -glucopyranoside or PNP-
b
-GlcNAc. The enzyme also
b
-
N
-acetyl-
D
-
What is the physiological role of Hex86 in Altero-
monas sp. strain O-7 ? Chitlaru and Roseman specu-
lated that ExoII cleaves phenolic b-GlcNAc deriva-
z
b
b
-
D
b
-
D
-
tives from chitin-producing organisms, such as fungi
and invertebrates, and the resulting phenol deriva-
xylopyranoside. To investigate whether Hex86 is in-
volved in the chitin degradation system of Alteromo-
nas sp. strain O-7, chitin oligosaccharides from
dimer to hexamer were used as a substrate. The assay
system for chitin oligosaccharides from dimer to
hexamer consisted of 0.1 ml each of enzyme solution,
1
6)
tives may induce V. furnissii to invade its host. On
the other hand, NagZ, the predicted amino acid
sequence of which is 57
hydrolyzed the
GlcNAc and anhydro-
z
identical to that of ExoII,
b
-1,4 glycosidic bond between
-acetylmuramic acid in cell
N
5
0 m
After incubation at 37
the GlcNAc produced was measured by the method
M
acetate buŠer (pH 5.5), and 5 m
M
substrate.
wall degradation products, suggesting that the
enzyme might participate in the cell wall recycling of
9
C for an appropriate period,
17)
E. coli
chitinases (ChiA, ChiB, and ChiC), a chitinase-like
enzyme (ChiD), and three -acetylglucosamini-
.
In Alteromonas sp. strain O-7, three
1
5)
of Reissig et al
was incubated with 28 mU of Hex86 at 37
0 min, there was no detectable GlcNAc in any of the
.
When each of the oligosaccharides
9
C for
b-N
1
dases (GlcNAcaseA, GlcNAcaseB, and GlcNAcaseC)
are involved in the chitinolytic system. However,
judging from the substrate speciˆcity of
reaction mixtures. Under the same assay conditions
as those for oligosaccharides, Hex86 rapidly hydro-
7
)
6)
lyzed PNP-
Hex86 prefers PNP-
rides as a substrate. Recently, ExoII of Vibrio furnis-
b
-GlcNAc. These results indicate that
Hex86, unlike GlcNAcaseA, GlcNAcaseB, and
5)
b
-GlcNAc to chitin oligosaccha-
GlcNAcaseC, the enzyme does not appear to take
part in the degradation of chitin oligosaccharides. To
clarify the physiological role of Hex86 in Alteromo-
nas sp. strain O-7, it remains to be investigated
whether the enzyme cleaves phenolic compounds
from chitin-producing organisms or cell wall degra-
dation products such as anhydro-muropeptides.
16)
17)
sii and NagZ of E. coli were shown to have an un-
usual substrate speciˆcity. These enzymes showed no
activity against chitin oligosaccharides, but rapidly
cleaved PNP-b-GlcNAc and slowly cleaved PNP-b-
GalNAc. Therefore, Hex86 has a similar substrate
speciˆcity with those of ExoII and NagZ under
the standard assay conditions described above.
However, after prolonged incubation (5 h) with
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3
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