Characterization of an α-L-rhamnosidase from Streptomyces avermitilis

Article abstract of DOI:10.1271/bbb.120735

The putative α-L-rhamnosidase gene from Streptomyces avermitilis was cloned and expressed. The recombinant enzyme released L-rhamnose from p-nitrophenyl α-L-rhamnoside, Citrus flavonoids such as naringin, rutin, and hesperidin, and gum arabic which is an

Full text of DOI:10.1271/bbb.120735

Biosci. Biotechnol. Biochem., 77 (1), 213–216, 2013
Note
Characterization of an ꢀ-L-Rhamnosidase from Streptomyces avermitilis
1;*
2
1;y
Hitomi ICHINOSE, Zui FUJIMOTO, and Satoshi KANEKO
1
Food Biotechnology Division, National Food Research Institute,
2
-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Biomolecular Research Unit, National Institute of Agrobiological Sciences,
2
2
-1-2 Kannondai, Tsukuba 305-8602, Japan
Received September 20, 2012; Accepted October 22, 2012; Online Publication, January 7, 2013
[doi:10.1271/bbb.120735]
The putative ꢀ-L-rhamnosidase gene from Streptomy-
ces avermitilis was cloned and expressed. The recombi-
nant enzyme released L-rhamnose from p-nitrophenyl
as a source of enzymes that degrade the sugar linkages
of AGPs. We cultivated this bacterium using gum
arabic, an AGP, as carbon source, and found that a ꢁ-L-
arabinopyranosidase (EC 3.2.1.88) and ꢀ-L-rhamno-
sidase (EC 3.2.1.40) are involved in AGP degrading
enzyme system of S. avermitilis (Fig. 1A). In the present
study, we focused on the characterization of a ꢀ-L-
rhamnosidase from S. avermitilis NBRC14893.
2
2)
ꢀ
-L-rhamnoside, Citrus flavonoids such as naringin,
rutin, and hesperidin, and gum arabic which is an
arabinogalactan-protein. Calcium ions increased L-
rhamnose production by the enzyme from gum arabic,
whereas enzyme activity was not affected by any metal
ions.
Because the complete genome sequence of S. aver-
3
2)
mitilis has been determined,
we knew how many
Key words: ꢀ-L-rhamnosidase; glycoside hydrolase fam-
ily 78; arabinogalactan-protein; Streptomy-
ces avermitilis
putative ꢀ-L-rhamnosidase genes the strain has. Glyco-
side hydrolases (GHs) are categorized into various
families in the Carbohydrate-Active enZymes (CAZy)
database according to similarity of amino acid
sequence. ꢀ-L-Rhamnosidases are classified into GH
families 28, 78, and 106. S. avermitilis possesses only
one putative ꢀ-L-rhamnosidase gene belonging to GH
family 78 (GH78), and does not possess any genes
belonging to GH families 28 or 106.
3
3)
Arabinogalactan proteins (AGPs) are a family of
proteoglycans that are localized on the cell surfaces of
higher plants. They play important roles in root
formation, the promotion of somatic embryogenesis,
1
)
and the attachment of pollen tubes. AGPs are charac-
terized by the presence of large amounts of carbohy-
drates, such as D-galactose and L-arabinose, and the
amino acids hydroxyproline, serine, threonine, alanine
and glycine.2 Type II arabinogalactans and short
oligosaccharides are two types of carbohydrate that are
attached to the AGP backbone. Type II arabinogalactans
have ꢁ-1,3-linked D-galactosyl backbones in mono- or
oligo-ꢁ-1,6-D-galactosyl and/or L-arabinosyl side
chains. L-Arabinose and lesser amounts of other auxil-
iary sugars, such as D-glucuronic acid, L-rhamnose, and
L-fucose, are attached to the side chains primarily at the
The full-length gene (3,090 bp) encoding a putative
GH78 ꢀ-L-rhamnosidase (sav 828; GenBank accession
no. BAC68538) was amplified from S. avermitilis
genomic DNA by polymerase chain reaction using the
)
0
following primers: forward, 5 -CAT ATG AGT GCC
0
0
TTG CGA GTC ACC TCT-3 ; and reverse, 5 -
0
AAG CTT AAC GGT GAA TCG GTG GCT TCC-3 .
The amplified DNA was cloned into pET30 vector
(Novagen, Madison, WI) at the NdeI and HindIII
restriction enzyme sites (underlined). A recombinant
protein, SaRha78A, was successfully expressed using
the T7 expression system in Escherichia coli Tuner
(DE3) (Merck KGaA, Darmstadt, Germany). The
protein was purified with a C-terminal histidine tag,
and was dialyzed against 50 mM phosphate buffer,
pH 7.0. The final preparation thus obtained was used
as purified enzyme. Protein amounts were determined
with the BCA protein assay reagent kit (Thermo
Scientific, Rockford, IL, USA) using bovine serum
albumin (BSA) as standard. The molecular mass of
SaRha78A on sodium dodecyl sulphate-polyacryamide
gel electrophoresis was estimated to be 113 kDa
(Fig. 1B, lane 3).
3
)
nonreducing termini. The complexity of AGPs com-
plicates the structural and functional analysis of indi-
vidual AGPs.
We are studying glycoside hydrolases that degrade the
–31)
4
carbohydrate moieties of AGPs.
We have first
cloned two key enzymes, exo-ꢁ-1,3-galactanase and
endo-ꢁ-1,6-galactanase, which hydrolyze the backbones
1
1,12)
of the AGP sugars.
In the course of research on
AGP-degrading enzymes, we found that Streptomyces
avermitilis NBRC14893 possesses both an exo-ꢁ-1,3-
1
6,17)
galactanase and an endo-ꢁ-1,6-galactanase.
Because
these enzymes work more effectively when sugars such
as L-arabinose and L-rhamnose are removed from the
galactan backbone, we have focused on this bacterium
The recombinant protein showed the p-nitrophenyl-
ꢀ-L-rhamnopyranoside (PNP-ꢀ-L-Rhap) hydrolyzing
y
*
To whom correspondence should be addressed. Fax: +81-29-838-7996; E-mail: sakaneko@affrc.go.jp
Present address: Applied Bacteriology Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Abbreviations: AGPs, arabinogalactan-proteins; BSA, bovine serum albumin; CAZy, Carbohydrate-Active enZymes; GHs, glycoside hydrolases;
GH78, glycoside hydrolase family 78; HPAEC-PAD, high-performance anion-exchange chromatography with a pulsed amperometric detection
system; PNP, p-nitrophenol; PNP-ꢀ-L-Rhap, p-nitrophenyl-ꢀ-L-rhamnopyranoside; SaRha78A, Streptomyces avermitilis ꢀ-L-rhamnosidase

214
H. ICHINOSE et al.
A
B
Rha
Ara
C
Rha Ara
Gal
Gal
Standards
Time (min)
Rha
Time (min)
Fig. 1. Hydrolysis Product of Gum Arabic and Expression of the Recombinant Protein.
A, HPAEC-PAD analysis of the hydrolysis products of gum arabic by culture supernatant of S. avermitilis cultivated with gum arabic. Rha,
rhamonose; Ara, arabinose; Gal, galactose. B, Sodium dodecyl sulphate-polyacryamide gel electrophoresis analysis of recombinant SaRha78A.
Lanes 1 and 2, molecular mass marker; lane 3, recombinant SaRha78A. C, HPAEC-PAD analysis of the hydrolysis product of gum arabic
generated by SaRha78A. Upper panel, standards (0.0002%); bottom panel, hydrolysis products of gum arabic by SaRha78A. Rha, rhamonose;
Ara, arabinose; Gal, galactose.
activity, but did not hydrolyze PNP-ꢀ-L-arabinofurano-
A
B
side, PNP-ꢀ-L-arabinopyranoside, PNP-ꢁ-L-arabinopy-
ranoside, PNP-ꢀ-L-fucopyranoside, PNP-ꢁ-D-fucopyra-
noside, PNP-ꢀ-D-galactopyranoside, PNP-ꢁ-D-galacto-
pyranoside,
PNP-ꢀ-D-glucopyranoside,
PNP-ꢁ-D-
glucopyranoside, PNP-ꢀ-D-xylopyranoside, PNP-ꢁ-D-
xylopyranoside, PNP-ꢀ-D-mannopyranoside, PNP-ꢁ-D-
mannopyranoside, PNP-ꢁ-D-glucuronide, or PNP-ꢁ-D-
galacturonide, suggesting that SaRha78A is an
ꢀ
tected at pH 6.0 at 50 C (Fig. 2A and B), and enzyme
-L-rhamnosidase. Maximal enzyme activity was de-
ꢀ
pH
Temperature (°C)
ꢀ
was stable between pH 5.0 and 11.0 at 30 C for 1 h, and
ꢀ
C
D
also stable below 40 C at pH 6.0 for 1 h (Fig. 2C and
D). The specific activity of SaRha78A with PNP-ꢀ-L-
Rhap as substrate was 7:6 ꢁ 0:2 units/mg at pH 6.0 at
ꢀ
5
0 C (Table 1). The kinetic parameters of SaRha78A
for PNP-ꢀ-L-Rhap were determined by the following
method: The reactions were performed in McIlvaine
buffer (0.2 M Na HPO and 0.1 M citric acid), pH 6.0,
2
4
containing 0.3–2 mM substrates, 0.1% (w/v) BSA, and
ꢀ
1
.2 mM enzyme at 30 C for up to 30 min. The K and
m
kcat values of SaRha78A for PNP-ꢀ-L-Rhap were 0:03 ꢁ
pH
Temperature (°C)
ꢂ1
0
:00 mM and 1:93 ꢁ 0:01 s respectively (Table 1).
The effects of metal ions and EDTA on enzyme
activity were examined by treatment with 2-mM solu-
tions of metal ions, including Ca , Cd , Co , Cu ,
Cs , Fe , Mg , Mn , and Zn and EDTA. The
metal ions and EDTA did not affect enzyme activity
when PNP-ꢀ-L-Rhap was used as substrate (data not
shown).
Fig. 2. Enzymatic Properties of Recombinant SaRha78A.
The effects of pH and temperature on enzyme activity and
stability were investigated. A, Optimum pH; B, optimum temper-
ature; C, stability for pH; D, stability for temperature. Symbols:
2
þ
2þ
2þ
2þ
2
þ
2þ
2þ
2þ
2þ
, McIlvaine buffer; , Atkins-Pantin buffer; , Glycine-NaOH
buffer.
activity toward an arabinogalactan-protein was deter-
ꢀ
The substrate specificities of SaRha78A were deter-
mined. The activities of SaRha78A toward flavonoids
such as naringin, rutin, and quercitrin were analyzed by
mined at 40 C in McIlvaine buffer (pH 6.0) with 2%
(w/v) gum arabic (Wako Pure Chemical Industries,
Osaka, Japan) as substrate and 1.8 mM enzyme. After
incubation for 24 h, the amount of L-rhamnose released
3
4)
the method described by Koseki et al. In contrast, the

ꢀ-L-Rhamnosidase from Streptomyces avermitilis
215
Table 1. Substrate Specificity of SaRha78A
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ꢀ-1
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19:5 ꢁ 1:1
0:9 ꢁ 0:07
28:5 ꢁ 2:9
N.D.
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ratio of L-rhamnose, L-arabinose, D-galactose, and D-
glucuronic acid was 5:20:28:6.
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activity for rutin (28:5 ꢁ 2:9 units/mg) and naringin
1
(
19:5 ꢁ 1:1 units/mg). It also showed activity for hes-
1
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than that for PNP-ꢀ-L-Rha, which was caused by the
difference between aglycon sides of the substrates. This
suggests that SaRha78A possesses subsite þ1, distin-
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Aspergillus aculeatus. SaRha78A cleaved both the
-1,6 and the ꢀ-1,2-linked rhamnosyl residues. These
activities have been reported for both bacterial and
fungal enzymes belonging to GH78.
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ꢀ
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reaction mixture. These results suggest that SaRha78A
has a region influenced by calcium ions, except for the
catalytic domain, because enzyme activity was not
affected by any of the metal ions (data not shown).
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(
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The present study indicates that S. avermitilis pos-
sesses a ꢀ-L-rhamnosidase acting on arabinogalactan-
proteins. Calcium ions increased the amount of L-
rhamnose produced from gum arabic by the enzyme. It
remains to be determined which region of the enzyme is
affected by calcium ions. A detailed structure-function
study would be expected to answer the function of
calcium ions in the arabinogalactan degradation by
SaRha78A.
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3
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This work was supported in part by JSPS KAKENHI
Grant no. 22580110. We thank Ms. Mariko Honda for
assistance in characterization of the recombinant enzyme.
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