An exo-β-(1→3)-d-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure

Article abstract of DOI:10.1016/j.carres.2012.02.033

An exo-β-(1→3)-d-galactanase (SGalase1) that specifically cleaves the β-(1→3)-d-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ～45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ～7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed β-(1→3)-d- galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The K _m and V_max of the native exo-β-(1→3)-d- galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 μmol d-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal₂GlcA₁ predominating.

Full text of DOI:10.1016/j.carres.2012.02.033

Carbohydrate Research 352 (2012) 70–81
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
An exo-b-(1?3)-D-galactanase from Streptomyces sp. provides insights into type
II arabinogalactan structure
Naomi X.-Y. Ling a, Joanne Lee a, Miriam Ellis a, Ming-Long Liao a, Shaio-Lim Mau a, David Guest c,
d
ˇ e
Peter H. Janssen , Pavol Kovác , Antony Bacic a,b, Filomena A. Pettolino a,
⇑
a Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Victoria 3010, Australia
b ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Victoria 3010, Australia
c Faculty of Agriculture, Food and Natural Resources, Biomedical Building C81, The University of Sydney, Eveleigh, NSW 2015, Australia
d Grasslands Research Centre, AgResearch Ltd, Tennent Drive, Private Bag 11008, Palmerston North 4442, New Zealand
e Laboratory of Bioorganic Chemistry, Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD 20892-0815, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An exo-b-(1?3)-D-galactanase (SGalase1) that specifically cleaves the b-(1?3)-D-galactan backbone of
Received 21 December 2011
Received in revised form 21 February 2012
Accepted 28 February 2012
Available online 8 March 2012
arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal pep-
tide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular
mass of the isolated enzyme was ꢀ45 kDa, similar to the 48.2 kDa mass predicted from the amino acid
sequence. The pI, pH and temperature optima for the enzyme were ꢀ7.45, 3.8 and 48 °C, respectively.
The native and recombinant enzymes specifically hydrolysed b-(1?3)-D-galacto-oligo- or poly-saccha-
Keywords:
b-D-Galactanases
Arabinogalactan-protein
Streptomyces sp.
De-arabinosylated gum arabic
CAZy family GH 43
rides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous
Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa)
and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2
are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homol-
ogy and phylogenetic analysis. The Km and Vmax of the native exo-b-(1?3)-
D-galactanase for de-arabi-
nosylated gum arabic (dGA) were 19 mg/ml and 9.7 mol -Gal/min/mg protein, respectively. The
l
D
activity of these enzymes is well suited for the study of type II galactan structures and provides an impor-
tant tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA)
was used as a model to investigate the use of these enzymes in defining type II galactan structure.
Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccha-
ride of Gal2GlcA1 predominating.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
plants, for example Arabidopsis,8 gum arabic (GA),9–11 gum traga-
canth,12 larch,13 apple,14 grape,15 radish16,17 and ryegrass.18 Re-
Arabinogalactan-proteins (AGPs) are a class of plant proteogly-
cans containing 90–99% (w/w) carbohydrate and 1–10% (w/w)
protein. The predominant carbohydrate components of AGPs are
type II arabinogalactans (AGs)1 together with some short oligoara-
binosides.2,3 Type II AGs consist of a highly branched and complex
cently, the Hyp-contiguity hypothesis has also contributed to the
better understanding of the glycosylation pattern of the protein
backbones of AGPs. The Hyp-contiguity hypothesis predicts that
non-contiguous Hyp residues would be exclusively substituted
with AG polysaccharide chains, while contiguous Hyp residues
would be glycosylated with oligoarabinosides.19–22 This hypothesis
was generated by studying heterologously-expressed synthetic
AGP protein backbones in tobacco and Arabidopsis.23 Hyp glycosyl-
ation may be tissue-specific and vary between species.22–24
Defining the precise structure of the carbohydrate moiety of
AGPs is essential in furthering our understanding of their biosynthe-
sis, as well as their biological and industrial functions. AGPs are pro-
posed to be responsible for a range of functions in plants.7 For
example, AGPs are proposed to play a role in signalling,25,26 embryo-
genesis,27,28 xylem differentiation,26,29 as well as cell expansion and
extension of apical tip growth in the moss Physcomitrella patens.30
The general structure of type II AG is well known, but its glycosyl
framework of a b-(1?3)-
galactopyranosyl (Galp) side chains, which are further substituted
with other sugars such as -L-arabinofuranose/pyranose (Araf/p),
D-galactan backbone and b-(1?6)-D-
a
glucopyranosyluronic acid (GlcpA), 4-O-methylglucopyranosyl-
uronic acid (4-O-MeGlcpA), L-rhamnopyranose (L-Rhap) or L-fuco-
pyranose (L-Fucp).4–7 Numerous studies have investigated the
structures of the carbohydrate component of AGPs from various
⇑
Corresponding author at present address. CSIRO Plant Industry, Black Mountain
Laboratories, Canberra ACT 2601, Australia. Tel.: +61 02 62464052; fax: +61 02
62465000.
E-mail address: Filomena.Pettolino@csiro.au (F.A. Pettolino).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.02.033

N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
71
sequences are yet to be elucidated, particularly the distribution and
degree of polymerisation (DP) of (1?3)- and (1?6)-linked Galp
chains.31 Chemical methods such as partial acid hydrolysis, alkaline
degradation, acetolysis and Smith degradation have been invaluable
in the analysis of the type II glycan chains of AGPs.15,18,32 However,
due to the complexity of the carbohydrate moiety, chemical ap-
proaches alone are not sufficient.33 Therefore, specific enzymes,
yield and the overall purification factor of the b-galactanase
achieved in the current study were 3.3% and ꢀ2-fold, respectively
(Table 1). The enriched b-galactanase fraction, HW 55 (major
peak), consisted of one major band at ꢀ45 kDa, one minor band
at ꢀ66 kDa and three other protein bands between 25 and
40 kDa in very low abundance (Fig. 2, Lane 5).
such as
ases (EC 3.2.1.31),35 and b-
1,3-
a
-L-arabinofuranosidases (EC 3.2.1.55),14,15,34 b-glucuronid-
-galactosidases (EC 3.2.1.23),34,36 exo-b-
-galactanases (EC 3.2.1.145)32,37 and endo-b-(1?6)-
D-galac-
2.2. Galactanase cloning and heterologous expression
D
D
The ꢀ45 kDa protein band was excised from the gel for N-
terminal sequencing and internal sequencing post in-gel trypsin
digestion. The N-terminal peptide sequence (A S A S F T L G A T Y
T D Q N) (Fig. 2 Lane 5) was compared to the NCBI non-redundant
protein databases using the BLASTp algorithm. Thorough searches
for short, nearly exact matches found no significant matches. How-
ever, the first 10 amino acids of the N-terminal peptide sequence
were the same as one of five trypsin generated peptides (high-
lighted in Fig. 3). The de novo amino acid sequences of the remain-
ing four trypsin-generated peptides (also in Fig. 3) from the same
protein band had a relatively high similarity to a number of proteins
across different regions of the amino acid sequences, with the top
tanases (EC 3.2.1.164)31,38,39 have been used to complement the
chemical methods. Most of these enzymes are not commercially
available and as a result, have only been used in a limited number
of studies. This is especially true of the galactanases that cleave b-
(1?3)- or (1?6)-
Furthermore, there is only one report to date of the isolation of an
endo-b-(1?3)-
-galactanase.40 Enzymes may have different modes
D-Galp linkages in either an exo- or endo-manner.
D
of action and/or catalytic sites. It is therefore important to isolate a
wide spectrum of hydrolytic enzymes that cleave specific glycosyl
linkages of the type II AGs. In the current study, an exo-b-(1?3)-D-
galactanase was isolated from culture filtrates of a Streptomyces
sp. isolated from soil and the kinetic properties of the enzyme deter-
mined. This enzyme, and a second similar enzyme from the same
source, were cloned and expressed in E. coli and their activity
characterised. We also demonstrate how the activity of these en-
zymes is well suited for the study of type II galactan structure.
hit in the NCBI BLAST search being exo-b-(1?3)-D-galactanase se-
quences from the CAZy database (http://www.cazy.org/CAZY).
These amino acid sequences were from Phanerochaete chrysospori-
um galactan (1?3)-b-galactosidase (Pc1,3Gal43A, gi 63108312),
Coprinopsis cinerea hypothetical protein (CC1G_03773, gi
116510091), Streptomyces avermitilis exo-b-(1?3)-
(Sal1,3Gal43A, gi 29828651) and Clostridium thermocellum exo-
b-(1?3)- -galactanase (Ct1,3Gal43A, gi 67916108). Commonly
conserved features of these sequences include regions showing se-
quence similarity to CAZy glycosyl hydrolase family 43 (GH 43) and
ricin carbohydrate-binding domains.
D-galactanase
2. Results
D
2.1. Native enzyme purification
Isolate 19 was selected from 26 cultures for its ability to grow on
medium supplemented with dGA and secrete protein with b-galac-
tanase activity. The 16S rRNA gene sequence revealed isolate 19 to
be a Streptomyces sp. related to a clade of species including
S. achromogenes, S. acidiscabies, S. antibioticus, S. bobili, S. coelicolor,
S. cyaneus, S. flavovariabilis, S. galilaeus, S. griseochromogenes,
S. griseoruber, S. longisporus, S. mirabilis, and S. phaeofaciens (data
not shown).
The culture filtrate (day 3) of Streptomyces sp. isolate 19 was
used to obtain b-galactanase by precipitating the proteins using
ammonium sulfate (crude enzyme preparation) followed by ion
exchange and gel filtration chromatography (Table 1). Most of
Using degenerate primers based on the N-terminal and internal
peptide sequences of the isolated protein, the SGalase1 gene se-
quence was amplified and sequenced, along with another putative
Streptomyces sp. galactanase gene, SGalase2. The predicted proteins
encoded by the two genes share 85% amino acid sequence similar-
ity (using mature protein sequence only) and a predicted size of
ꢀ48 kDa for the mature enzymes (Fig. 3). SGalase2 has a predicted
N-terminal signal peptide and conserved domain searches41 found
that both of the predicted proteins contain a GH 43 catalytic do-
main at the N-terminal end, and a ricin-type b-trefoil carbohy-
drate-binding domain at the C-terminal end (Fig. 3). Despite
repeated attempts, the very 50 end of SGalase 1 proved difficult to
obtain, however based on protein sequence analysis, the majority
of the enzyme was cloned. Sequences for SGalase 1 and SGalase
2 were deposited in Genbank with accession numbers JQ683399
and JQ683400 respectively.
The amino acid sequences for the catalytic domains of the
characterised GH43 enzymes were aligned with those of SGalase1
and SGalase2, and the program MEGA 5 was used to generate a
Neighbor-Joining tree (Fig. 4). The tree shows, with high bootstrap
support, that the two SGalase enzymes are more similar to each
other than to any of the other enzymes, and that they are nested
within the other exo-b-(1?3)-galactanases.
the unwanted b-
D
-galactosidase activity (ꢀ90%) in the crude en-
zyme preparation was removed by binding to DEAE Trisacryl (M)
at pH 8 (data not shown). The remaining DEAE unbound fraction
separated into four major protein peaks by cation exchange chro-
matography (Fig. 1). Most (89%) of the b-galactanase activity eluted
as a single peak (EMD Peak 2, Fig. 1) in the range of 0.1–0.15 M
NaCl, while most of the
a-L-arabinofuranosidase and remaining
b- -galactosidase activities eluted at or above 0.75 M NaCl, well
D
separated from the region associated with b-galactanase activity.
EMD Peak 2 was further fractionated by size on a TSK HW 55 col-
umn and the b-galactanase activity eluted as a major peak, corre-
sponding to a molecular mass of ꢀ43 kDa (data not shown). The
Table 1
Protein content and enzyme activity of different fractions obtained in the purification of b-galactanase from filtrate of 3 day-old culture of Streptomyces sp.
Fraction
Total protein
(mg)
Protein
yield (%)
Total activitya
(Units)
Specific activity
(Units/mg protein)
Yield
factor (%)
Purification
(fold)
(NH4)2SO4 ppt (Crude enzyme)
DEAE Trisacryl (Unbound)
Fractogel EMD SO3 (EMD Peak 2)
323
111
7
100
34
2.2
1.8
6975
3720
279
22
34
40
40
100
53
4
1
1.5
1.8
1.8
ꢁ
Fractogel TSK HW 55 (Major peak)
5.86
233
3.3
a
Units = One unit (U) of b-galactanase activity is defined as the amount of enzyme that liberates 1 nmol of low molecular weight substrate fragments bound with RB5 dye
per minute under the assay conditions described.

72
N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
ꢁ
Figure 1. Elution profile of protein and enzyme activities on Fractogel EMD SO3 resin. b-Galactanase activity was determined as described in the methods, except a different
amount of sample (50
b-galactosidase activity (s; U/10) and
lL of fraction collected) and a longer incubation (1.5 h) were used. Absorbance at 280 nm (—), NaCl concentration (.....), b-galactanase activity (4; Ux3),
a
-arabinofuranosidase activity (d; U/200). The pooled fractions are shaded (58–68 min).
2.3. Enzyme characterisation
The pI of the isolated Streptomyces sp. b-galactanase was ꢀ7.45
kDa
116
as determined by IEF gel electrophoresis and had a pH optimum of
3.8 at 37 °C, and temperature optimum of 47.5 °C at pH 3. The pH
optima of recombinant SGalase1 and SGalase2 were 3.8 and 5.0 at
37 °C, respectively. The temperature optimum of both recombinant
enzymes was 48 °C at their appropriate pH optima. All enzymes
were most stable up to 37 °C.
66.2
45
35
Each of the enzymes was tested against a broad panel of oligo-
and poly-saccharides (Supplementary Table 1) and were found to
be active only against oligo- and poly-saccharides containing b-
25
(1?3)-
strates, as well as the percentage (%) relative to the specific activity
towards a ‘b-(1?3)- -galactan’ (Acacia) preparation, are presented
in Table 2. The enzymes had no, or negligible, activity on p-
nitrophenyl (PNP) b- -galactopyranoside, PNP b- -fucopyranoside,
PNP -L-arabinofuranosidase PNP -galactopyranoside, PNP b/
-glucopyranoside, PNP b/ -L-fucopyranoside, PNP b-
cellobioside, PNP -mannopyranoside or PNP b- -xylopyranoside
after 24 h incubation at 37 °C.
D-Gal linkages. The specific activities against these sub-
18.4
D
14.4
D
D
a
a-D
a-D
a
D-
a-D
D
2
3
4
5
1
All three enzyme preparations had similar substrate specificity,
but the native enzyme and its recombinant form, SGalase1,
had higher specific activities than SGalase2. Among the methyl b-
Figure 2. SDS–PAGE of various protein fractions obtained during the purification
process. Lanes: (1) Molecular weight markers, (2) crude enzyme, (3) DEAE Trisacryl
ꢁ
unbound fraction, (4) Fractogel EMD SO3 bound fraction (EMD Peak 2; Figure 1),
glycosides of b-(1?3)-linked D-galacto-oligosaccharides tested, the
and (5) Fractogel TSK HW 55 (major peak; Figure 2).
specific activity of the enzyme was highest towards the galactobio-
side. The specific activities of the enzymes decreased dramatically
at DP 3 and remained relatively similar across DP 3–5 and 7. The
SGalase 1 and SGalase 2 were cloned into E. coli and expressed.
Although both proteins formed protein inclusion bodies, their re-
natured forms (after urea solubilisation) retained galactanase
activity, appeared as single protein bands by SDS–PAGE and were
therefore used for further characterisation.
enzymes hydrolysed the ‘b-(1?3)-
rates compared to the methyl b-glycosides of b-(1?3)-linked
galacto-oligosaccharides. The specific activity of the SGalase1 and
2 towards dGA were only 30% and 37%, respectively, of that towards
the ‘b-(1?3)-D-galactan’. The kinetic parameters of the enzyme
D-galactan’ and dGA at lower
D-

N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
73
Figure 3. Alignment of SGalase1 and SGalase2 amino acid sequences. Conserved amino acids are highlighted in black, predicted SGalase2 signal peptide is boxed, catalytic
domain is underlined and carbohydrate-binding domain is double-underlined. SGalase1 peptides used to design degenerate primers are indicated by grey lines above the
sequences. Numbers on the right indicate amino acid position.
towards dGA were determined by a Lineweaver–Burk plot from
which Km = 19 mg/ml and Vmax = 9.7 mol -Gal/min/mg protein
were obtained. Native AGPs from Acacia, the AG from larch (Larix),
b-(1?3;1?6)-galactan from P. zopfii and the methyl b-glycoside of
b-(1?6)-linked D-galacto-oligosaccharides were resistant to hydro-
age analysis, with a higher amount in the enzyme hydrolysed dGA
ppt fraction (Table 3). However, no Glcp was detected in the mono-
saccharide composition analysis (by methanolysis) in any of the
samples, suggesting Glcp is likely to be a contaminant sugar intro-
duced during the methylation analysis.
l
D
lysis by the enzymes after extended incubation of 24 h (Table 1).
The modes of action of SGalase1 and SGalase2 were studied by
The linkage analysis for neutral sugars of the dGA showed that
it was composed mainly of Galp (ꢀ68 mol %), with terminal
(5 mol %), 1,3-linked (3 mol %), 1,6-linked (34 mol %) and 1,3,6-
linked (26 mol %) (Table 3). Compared to the dGA, the enzyme
hydrolysed dGA had increases in terminal Galp (from 5 to 16 and
33 mol % for ppt and supt fractions, respectively), and 1,6-linked
Galp (from 34 to 40 and 54 mol % for supt fraction, respectively).
Notably, a significant decrease in 1,3,6-linked Galp for both ppt
and supt fractions (from 26 to 18 and 1 mol %, for ppt and supt
fractions, respectively) was observed. Moreover, 1,3-linked Galp
was absent in the enzyme hydrolysed dGA supt fraction, while it
remained relatively unchanged in the ppt fraction (Table 3). No su-
gar was detected in the control dGA supt fraction, while the linkage
composition of the control dGA ppt fraction was essentially the
same as that of the untreated dGA (Table 3).
The MALDI-TOF MS spectrum of the final enzyme product after
exhaustive hydrolysis displayed 6 major molecular ion species of
m/z 673.2, 701.2, 919.2, 961.2, 1149.2 and 1191.2 (Fig. 6 and Table
4). These ions were present in replicate experiments and in each case
the m/z 961 ion dominated. The deduced structure of each ion was as
follows: (1) peracetylated Hex1HexA1 (m/z 673.2), (2) peracetylated
Hex2 (m/z 701.2), (3) peracetylated Hex1HexA2 (m/z 933.2), (4) per-
acetylated Hex2HexA1 (m/z 961.2), (5) peracetylated Hex2HexA1-
Pent (m/z 1177.2), and (6) peracetylated Hex2HexA1DeoxyHex1
(m/z 1191.2), as the sodium adducts. The peaks at m/z 919.2 and
1149.2 represent underacetylated [minus an acetyl (m/z 42) group]
species of m/z 961.2 and 1191.2, respectively. The peak at m/z 977.1
represents the K+ adduct of the major ion, peracetylated Hex2HexA1,
and it should be noted that the 1149.2ion may also represent the Na+
adduct of Hex1HexA2Pent. Peaks at 605, 956 and 983 (m/z) were
unidentified.
TLC (Fig. 5). Hydrolysates of the b-(1?3)-linked
anoside showed the release of Gal/methyl b-glycosides of b-(1?3)-
linked -galacto-oligopyranosides with DP lower than DP 4. Nota-
bly, methyl b- -galactopyranoside (GalpOMe) was not observed
D-galactotetrapyr-
D
D
initially. As the incubation progressed, Gal and/or Galp2OMe
(Gal and Galp2OMe were not well resolved on the TLC) and
GalpOMe continued to accumulate with the concomitant disap-
pearance of the higher oligomers. The final hydrolysis products
showed two major spots on the TLC plate, corresponding to Gal-
pOMe, and Gal and/or Galp2OMe. The Gal and Galp2OMe may be
better resolved by modification of solvent conditions or alterna-
tively, identified by mass spectrometry. Nevertheless, the absence
of GalpOMe at the initial stages of hydrolysis, demonstrated that
SGalase1 and SGalase2 released Gal from the upstream end of b-
(1?3)-linked
D-galactotetraopyranosides (the non-reducing end
in the non-methylated equivalent), thus catalysing the hydrolysis
in an exo-manner.
dGA was treated with enzyme and then ethanol precipitated to
separate the high molecular weight (ppt fraction) from the low
molecular weight (supt fraction) components, to study the struc-
tures of the products of enzyme digestion. The monosaccharide
composition of the dGA (starting material, Table 3) determined
by methanolysis showed that the most abundant sugar was Gal
(73%), followed by a significant amount of GlcA (25%), and low
levels of Rha (2%) and Ara (<0.5%). The monosaccharide composi-
tions of enzyme hydrolysed dGA ppt and supt fractions were sim-
ilar in Gal (61% and 65%, respectively) and Rha (4% and 2%,
respectively), and both had 34% GlcA, and a minimal amount of
Ara (1%). A low level of Glc was detected in all the samples in link-

74
N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
Bp CAA29235
Bp AAC97375
BSp O52729
Bs NP 389640
92
91
β-xylosidase
97
91
95
Bst ABI49959
Sr AAB97967
VSp BAF98235
86
Xylosidase/Arabinosidase
β-(1,3)-xylosidase
85
Xylosidase/Arabinosidase
Xylosidase/Arabinosidase
Arabinofuranosidase
Bf AAA63610
ub ACF39706
ub ABB92159
Cs CAD48310
Cs BAC87941
Ba AAO67499
Bs ACB06752
Bs NP 390759
Bt BAB64339
Cj CAA71485
And AN6352.2
And AN8007.2
Aa AAG27441
An AAA32682
Cs BAA02527
Sc BAA90772
Pb CAA89208
Ph BAC75546
Cc AAC67554
Bs Q45071
31
59
100
83
Arabinofuranosidase
100
β-xylosidase
Arabinoxylan arabinofuranohydrolase
61
65
97
89
Endo-(1,5)-arabinase
93
87
69
99
Xylosidase/Arabinosidase
45
Exo-(1,5)-arabinofuranosidase
99
100
β-xylosidase
62
Arabinoxylan arabinofuranohydrolase
Xylosidase/Arabinosidase
100
Pp CAA40378
Ct ABN51896
Sa BAC69820
SGalase1
30
93
100
100
Exo-β-(1,3)-galactanase
SGalase2
87
Il BAH29957
Pc BAD98241
100
0.1
Figure 4. Phylogenetic tree of the catalytic domains of the SGalase proteins and characterised CAZy family 43 glycosyl hydrolases. The evolutionary history was inferred
using the Neighbor-Joining method. The optimal tree with the sum of branch length = 8.35 is shown. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (500 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary
distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid
differences per site.
overall purification factor of the Streptomyces sp. exo-b-(1?3)-D-
3. Discussion
galactanase (Table 1) was low compared to those reported for b-
galactanases in other studies (>12-fold).33,37,42 The reason for a
low purification factor is not fully understood, but could be attrib-
We have successfully purified, cloned and heterologously ex-
pressed an exo-b-(1?3)-D-galactanase from Streptomyces sp. The

N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
75
Table 2
Activity of the native, and recombinant SGalase 1 and SGalase 2 on b-(1?3)/(1?6)-D-linked polysaccharides and galacto-oligosaccharides
Substrate
Native enzyme
Specific activity SD
mol -Gal/min/mg
protein)
SGalase 1
Specific activity SD
mol -Gal/min/mg
protein)
SGalase 2
Specific activity SD
mol -Gal/min/mg
protein)
Relative
specific
activity (%)
Relative
specific
activity (%)
Relative
specific
activity (%)
(l
D
(l
D
(l
D
Polysaccharides
b-(1?3)-D-Galactan (Acacia)
10.8 0.3
4.4 0.1
0.03 0.0
0.05 0.0
0.1 0.0
100
20
ꢀ0
ꢀ0
1
12.4 0.4f,c
3.8 0.2e,g
0.0 0.0e,h
0.0 0.0e,h
0.0 0.0e,h
100
31
0
0
0
3.8 0.1f,c
1.4 0.1e,g
0.0 0.0e,h
0.0 0.0e,h
0.0 0.0e,h
100
37
0
0
0
dGA (Acacia)
Gum arabic (Acacia)
Arabinogalactan (Larch)
b-(1?3;1?6)-
Oligosaccharides
Methyl b-glycosides of b-(1?3)-
D
-Galactan (P. zopfii)
D-
Galactobiopyranoside
Galactotriopyranoside
63.2 1.6
nd
49.8 0.4
44.2
293
nd
230
205
nd
92.0 5.8a,b
28.5 3.9d,b
27.9 1.1d,b
32.6 6.0d,b
21.7 0.6d,b
740
229
225
262
175
37.10 1.9a,c
5.2 0.2e,c
4.2 0.1e,c
6.7 0.1e,c
3.4 0.3e,c
972
137
111
175
89
Galactotetrapyranoside
Galactopentaopyranoside
Galactoheptaopyranoside
Methyl b-glycosides of b-(1?6)-
Galactotriopyranoside
Galactotetraopyranoside
Galactopentaopyranoside
Galactohexaopyranoside
nd
D-
0.0 0.0
nd
0.2 0.0
0.2 0.0
0
nd
2
0.0 0.0e,h
0.0 0.0e,h
0.0 0.0e,h
0.0 0.0e,h
0
0
0
0
0.0 0.0e,h
0.0 0.0e,h
0.0 0.0e,h
0.0 0.0e,h
0
0
0
0
2
a
f
nd, not determined; Enzyme at 0.1, d0.5, e0.7, 0.4
l
g protein and incubated for b5, c15, g30 min, h24 h at 37 °C.
uted to the presence of several enzymes that all possess b-
galactanase activity within the crude enzyme as well as the nature
of the assay used. The total b-galactanase activity of the crude en-
zyme preparation may actually reflect the combined activities of
protein/mg galactan III/mL33 and 64.3
tein/mM ‘b-(1?3)-
-galactan’,37 and Kcat/Km values of 263,45
30842 and 175846 minꢁ1 mMꢁ1 ‘b-(1?3)-
-galactan’.
The activity profile observed for the Streptomyces sp. exo-b-
(1?3)- -galactanases, SGalase 1 and SGalase 2, towards different
substrates is distinct from the previously reported exo-(1?3)-
galactanases, which exhibit higher activities towards the ‘b-
(1?3)- -galactan’ than b-(1?3)- -galacto-oligosaccharides or
methyl b-glycosides of b-(1?3)-linked -galacto-oligosaccha-
lmol D-Gal/min/mg pro-
D
D
multiple enzymes, b-(1?3)- and/or b-(1?6)-D-galactanases and
D
their isozymes, rather than the activity of a single dominant
enzyme. Faint clearing zones were seen in zymograms with gels
containing the chromogenic substrate and run under native condi-
tions (data not shown).43 The clearing patterns of the crude and
final isolated enzyme fractions were different, suggesting that
the final preparation was not representative of all b-galactanase
activities in the crude preparation. Besides, the chromogenic sub-
strate (RB5-dGA) contains both b-(1?3)- and (1?6)-galactosyl
linkages, therefore precise measurement of a single b-galactanase
in the presence of multiple b-galactanases is challenging. The
purification fold could only be determined more accurately if
linkage-specific substrates were available.
D-
D
D
D
rides.37,42,46 The exo-b-(1?3)- -galactanases from A. niger37 and S.
D
avermitilis42 showed increasing activity towards b-(1?3)-galacto-
oligosaccharides and their methyl b-galactosides with increasing
DP from 2 to 5, while the exo-b-(1?3)-
sosporium46 had a higher activity towards b-(1?3)-
than towards b-(1?3)- -galactotriose. The exo-b-(1?3)-
galactanase from C. thermocellum exhibited a slightly higher
activity towards b-(1?3)- -galactotriose than towards the ‘b-
D-galactanase from P. chry-
D
-galactobiose
D
D-
D
The molecular mass of the Streptomyces sp. exo-b-(1?3)-
galactanase is within the range (38–66 kDa as estimated by SDS–
PAGE) of other exo-b-(1?3)-
-b-galactanases,33,37,42,44,45 while its
pI is lower than those from Irpex lacteus.37 The pH optima of most
other exo-b-(1?3)- -galactanases were reported within the range
of 4.5–6.33,37,44,42,45,46 Most of the b-(1?3)-
-galactanases appear
D
-
(1?3)-D D-galactanases reported thus
-galactan’.45 All the b-(1?3)-
far have high substrate specificity, except that from R. niveus,47
which hydrolysed coffee bean AG to liberate Ara, Gal and b-
D
(1?6)-
about the purity of the b-(1?3)-
The results on mode of action of the isolated enzyme are consistent
D
-galactobiose. However, questions have been raised
D
D
-galactanase from R. niveus.31,33,37
D
to be stable in acidic to neutral, but not alkaline conditions,33,37,42,46
except that from Clostridium thermocellum, which is stable in both
acidic and alkaline conditions, spanning a wide pH range of
3–10.44 The temperature optimum of the isolated and recombinant
with those reported for the exo-b-(1?3)-
D-galactanases from I.
lacteus,37
P. chrysosporium,46 C. thermocellum45 and S. avermitilis,60 where only
Gal was reported as the hydrolysis product released from the non-
enzymes is similar to that of previously identified exo-b-(1?3)-
D
-
reducing end of the ‘b-(1?3)-
After exhaustive hydrolysis of the dGA with the Streptomyces sp.
exo-b-(1?3)- -galactanase, the extent of hydrolysis was deter-
D
-galactan’.42,45,46
galactanases (40–50 °C).33,37,42,44
The Vmax/Km value of the native isolated enzyme for dGA
(0.5 mol -Gal/min/mg protein/mg dGA/mL) is relatively low, sug-
D
l
D
mined to be ꢀ20%. This is defined as the reducing sugars released
after extensive hydrolysis (equivalent to 1.2 mg Gal) relative to the
total sugar content (equivalent to 6 mg Gal) of the substrate used.
After ethanol precipitation of the extensively hydrolysed dGA, the
precipitate (ppt) and supernatant (supt) fractions accounted for
23% and 77% of the total sugar content, respectively (Table 3).
The extent of hydrolysis of dGA by Streptomyces sp. exo-b-(1?3)-
gesting dGA is unlikely to be the best substrate for the enzyme. This
is not surprising, since dGA is a highly branched glycan consisting of
26 mol % 1,3,6-linked Galp and only a low level of 1,3-linked Galp
(3 mol %).43,44 Based on the relative activity measured against
different substrates (see Table 2), it is likely that the Vmax/Km values
of this enzyme would be higher for methyl b-glycosides of b-(1?3)-
linked
ability of these synthetic substrates, the kinetic parameters could
not be further explored. Some exo-b-(1?3)- -galactanases were re-
ported to have Vmax/Km values of 43.6 mol galactobiose/min/mg
D
-galacto-oligosaccharides. However, due to limited avail-
D-galactanase is similar to that reported for exo-b-(1?3)-D-
galactanases from A. niger on galactan III (ꢀ18%),33 as well as that
D
achieved by the exo-b (1?3)-D-galactanase from I. lacteus on the a-
l
L-arabinofuranosidase-treated radish leaf AGP (21%).37 In addition,

76
N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
GalpOMe
Galp
Galp2OMe
Galp3OMe
Galp4OMe
1
2
3
4
5
6
7
8
9
10
11 12
13
Figure 5. Mode of action of the SGalase1 and 2 on methyl b-glycosides of b-(1?3)-linked
volume) contained 0.1 g enzyme and 1.4 L of methyl b-glycosides of b-(1?3)-linked -galactotetraopyranoside (Galp4OMe, 50 mM) and was incubated for 0 min (lane 6
and 10 for SGalase 1 and 2, respectively), 5 min (lane 7 and 11 for SGalase 1 and 2, respectively), 30 min (lane 8 and 12 for SGalase 1 and 2, respectively) or 3 h (lane 9 and 13
D-galactotetrapyranoside analysed by TLC. The reaction mixture (4.2 lL final
l
l
D
for SGalase 1 and 2, respectively) at 37 °C. Standards on lanes: (1) Galp4OMe, (2) methyl b-glycosides of b-(1?3)-linked D-galactotriopyranoside (Galp3OMe), (3) methyl b-
glycosides of b-(1?3)-linked
D-galactobiopyranoside (Galp2OMe), (4) methyl b-
D
-galactopyranoside (GalpOMe), (5) galactose (Galp). ⁄Galp and Galp2OMe virtually co-
migrated.
the b-galactosidase from radish48,49 and spinach34 were reported to
achieve 17–31% extent of hydrolysis of an -L-arabinofuranosi-
radish root AGP, leading to removal of 91% carbohydrate from
the substrate.37
a
dase-treated radish leaf AGP. Although the extent of hydrolysis
was modest, a substantial carbohydrate component of the dGA
was released as low DP glycosyl moieties after Streptomyces sp.
exo-b-(1?3)-D-galactanase hydrolysis, as indicated by the high
proportion (77%) of the total sugar content in the enzyme hydroly-
sed dGA supt fraction (Table 3). By comparison, the
The limited extent of hydrolysis of various substrates contain-
ing b-(1?3)-linked Gal residues is presumably due to the presence
of structure(s) that would interfere with the galactanases in gain-
ing access to the b-(1?3)-Gal linkages,37,49 such as the presence
of uronic acids at the non-reducing ends of b-(1?6)-linked Gal side
chains of most of the AGPs.6,7,49 Supporting this conclusion is the
reported increase in the extent of hydrolysis from ꢀ14% to ꢀ80%
exo-b-(1?3)-D-galactanase from I. lacteus was able to achieve
26% extent of hydrolysis of an
a-L-arabinofuranosidase-treated
when a-L-arabinofuranosidase-treated radish leaf AGP was hydro-
Table 3
Composition of the dGA and the Streptomyces sp. exo-b-(1?3)-D-galactanase hydrolysed dGA product
Sample
Starting material2
Enzyme hydrolysed dGA3
Control dGA (no enzyme)4
ppt supt
GA1
ND
dGA
ND
ppt
23
supt
77
Total sugar content5 (% SD)
5
8
100 10
0
0
Methanolysis6
Monosaccharide composition (weight %)
Rha
Ara
Gal
ND
ND
ND
ND
2
tr
73
25
4
1
61
34
2
1
65
34
ND
ND
ND
ND
ND
ND
ND
ND
GlcA
Linkage analysis7
Deduced glycosidic linkage
Linkage composition (mol%)
Rhap
Arap
Araf
Terminal
Terminal
Terminal
1,3-
Terminal
1,4-
Terminal
1,3-
1,6-
1,4,6-
1,3,4-
1,3,6-
1,2,6-
10
1
14
13
2
16
14
1
tr
—
1
18
—
2
4
2
2
1
5
1
2
2
ND
ND
33
—
54
1
—
1
1
4
—
—
—
—
ND
ND
—
—
—
—
—
—
—
—
—
—
—
—
tr
28
2
tr
tr
1
ND
ND
9
6
38
4
—
35
tr
1
tr
tr
GlcpA
Galp
ND
ND
16
4
40
1
—
18
1
tr
9
5
3
34
—
—
26
—
—
—
—
1,3,4,6-
1,4-
1,2,3,4,6-
10
—
—
tr
—
tr
Glcp
2
ND, not determined; —, not detected; tr, trace <1 mol %; ppt, precipitate fraction; supt, supernatant fraction.

N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
77
lysed by the combined action of radish b-galactosidase and a
microbial b-glucuronidase.49 The sugar composition data of dGA
used in the present study also showed the presence of high levels
of terminal uronic acids (Table 3), suggesting that future studies of
the extent of hydrolysis of dGA when treated simultaneously with
b-galactosidase and b-glucuronidase would be advisable to gener-
residue present in the enzyme hydrolysed dGA supt fraction
(Table 3). Therefore, the deoxyHexose containing oligosaccharide
must contain Rhap as the terminal residue.
No 4-O-MeGlcpA or any other monosaccharide residue was
evident from MALDI-TOF MS analyses (Fig. 6). This is not surprising
because there is only a very low level of 4-O-MeGlcpA in gum ara-
bic from A. senegal.51 As shown in Table 3, the enzyme hydrolysed
dGA supt fraction contained mainly terminal Galp (33 mol %) and
1,6-linked Galp (54 mol %), with depletion of both 1,3- and 1,3,6-
linked Galp; while the ppt fraction had a lower proportion of
1,3,6-linked Galp compared to the dGA. This suggested that Strep-
ate the preferred substrate for the Streptomyces sp. exo-b-(1?3)-D-
galactanase.
The enzymes isolated and cloned in this study show substrate
specificities similar to those of the exo-b-(1?3)-D-galactanases
that are also classified in CAZy GH 43.42,45,46 The CAZy database
(http://www.cazy.org/)50 classifies glycoyl hydrolases into 130
families based on amino acid sequence similarities. Recently,
tomyces sp. exo-b-(1?3)-D-galactanase was able to cleave the b-
1,3-linkages of both the 1,3- and 1,3,6-linked Galp residues, releas-
ing oligosaccharides of DP 2–4, made up of GlcpA-(1?6)-Galp and
Galp-(1?6)-Galp with or without GlcpA, Araf or Rhap attached
(MALDI-TOF MS data, Table 4). The rather small set of oligosaccha-
ride structures enzymatically released from de-arabinosylated
gum arabic AGPs in this study is consistent with the findings of
Tan et al.52 that proposed small repetitive subunits combining to
form the much larger AGs on AGPs.
Kotake et al.40 reported on the isolation of an endo-b-(1?3)-
D-
galactanase from the mushroom, Flammulina velutipes. Sequence
information and phylogenetic analysis determined that this en-
zyme belongs to CAZy GH 16, a family containing other endo-acting
enzymes, including endo-b-(1?3)- and endo-b-(1?3, 1?4)-
glucanases. GH 43 contains several hundred enzymes, including
xylosidases, arabinofuranosidases and galactanases. The galactan-
ases in GH 43, as well as the enzymes from the current study, show
only the galactan-b-(1?3)-galactosidase activity but not the activ-
ities of other enzymes reported for the GH 43 family, including b-
4. Experimental
4.1. Source of enzyme and culturing
xylosidase (EC 3.2.1.37),
a-L-arabinofuranosidase (EC 3.2.1.55),
endo- -(1?5)-L-arabinanase (EC 3.2.1.99) and b-(1?4)-xylanase
a
Dilutions of soil collected from a rotationally-grazed pasture at
the Dairy Research Institute, Ellinbank, Australia, were plated onto
plates of medium VL55 solidified with agar53 and containing 0.05%
(w/v) gum arabic. Twenty-six rapidly-developing colonies were
subcultured to purity, and then transferred to plates containing
0.05% (w/v) de-arabinosylated gum arabic (dGA). Isolate 19, which
grew well on dGA and secreted b-galactanases, was selected for
further investigation.
The isolate, identified as a Streptomyces species, was maintained
at 28–30 °C in the dark on a synthetic agar medium containing
MgSO4ꢂ7H2O (20 mM), CaCl2ꢂ2H2O (3 mM), (NH4)2HPO4 (20 mM),
KCl (5 mM), NaCl (10 mM), selenite/tungstate solution (1 mL/L),54
trace element SL10 solution (2 mL/L),55 MES (9.75 g/L), dGA (5 g/
L) and granulated agar (15 g/L), adjusted to pH 5.5. A hyphal plug
(0.5 cm diameter) was diced and added to medium (2 mL), pre-
pared as above (without agar) in a 12 mL sterile polypropylene
tube, and incubated at 30 °C with shaking (150 rpm). The culture
was bulked up by a series of 3 day subcultures until it was inocu-
lated (17.5 mL) into the liquid medium (350 mL) in a 1000 mL
flask. After 3 days, the culture was harvested by centrifugation
(3373xg, 10 min), and the supernatant filtered through Whatman
No. 541 filter paper to give the culture filtrate.
(EC 3.2.1.8) activities (http://www.cazy.org/fam/GH43.html). Phy-
logenetic analysis of the characterised GH 43 family members
showed that the galactanases and arabinanases each formed a
monophyletic group with strong bootstrap support. The xylosidas-
es generally formed two clades that are well separated from one
another, with one being closer to the endo-arabinanases and the
other closer to the galactanases. The dual-function xylosidase/
arabinofuranosidases and remaining arabinofuranosidases scatter
throughout the phylogenetic tree and do not appear to form func-
tional groups based on enzyme activity. The absence of clear phy-
logenetic grouping based on protein sequence alone demonstrates
that the characterisation of the activities of these enzymes remains
an important aspect in the study and use of glycosyl hydrolases. It
enables further organisation of the members of each family into
functional groups dependent on substrate specificity and poten-
tially highlights sequence information that will aid in identifying
particular enzyme activities at the transcript level.
Data of the hydrolysed dGA products from the current project
are similar to those reported for other exo-b-(1?3)-
D-galactanases
acting on AGPs. For example, the exo-b-(1?3)- -galactanases from
D
A. niger degraded galactans II and III (prepared by acid hydrolysis
and two successive Smith degradations of the gum arabic from A.
senegal), releasing Gal, b-(1?6)-
or without Ara attached, and other unidentified reaction products
of higher DP.33 The exo-b-(1?3)- -galactanases from I. lacteus,37
P. chrysosporium,46 C. thermocellum45 and S. avermitilis42 hydroly-
sed -L-arabinofuranosidase-treated radish root AGPs releasing
Gal and b-(1?6)- -galacto-oligosaccharides with or without 4-O-
MeGlcpA. In the current study, the released oligosaccharides were
analysed using MALDI-TOF MS. The assigned structures showed
the presence of a mixture of oligosaccharides in the enzyme
hydrolysed dGA 80% ethanol supt fraction. Several sequences are
possible, but the proposed oligosaccharide structures have been
deduced based on the following observations: (1) The reducing
D-galacto-oligosaccharides with
4.2. Substrates and sugar standards
D
Various p-nitrophenyl (PNP) glycosides and oligo-/poly-saccha-
rides, including methyl b-(1?3)-D-galactobioside, and a-(1?3)-D-
galactobiose were purchased from Sigma–Aldrich (St. Louis,
Missouri, USA), Megazyme (Bray, Co. Wicklow, Ireland), Applichem
Inc. (Cheshire, CT, USA), or Biosupplies (Melbourne, Australia). ‘b-
a
D
(1?3)-D-Galactan’ (Acacia senegal), a Smith degraded product of
gum arabic (GA) with an estimated DP of 50,46 and b-
(1?3;1?6)-galactan from Prototheaca zopfii with molecular mass
of 85 kDa38 were generous gifts from Professor Y. Tsumuraya
(Department of Biochemistry and Molecular Biology, Faculty of Sci-
ence, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama
338-8570,
end sugar of the Streptomyces sp. exo-b-(1?3)-D-galactanase
hydrolysed dGA must be Galp since the enzyme specifically cleaves
only 1,3-Galp. (2) Most of the GlcpA in the dGA were terminal res-
idues (28 mol %), with only 2 mol % 1,4-linked (Table 3). Therefore,
the HexA-containing oligosaccharides predominantly consist of
terminal GlcpA rather than 1,4-linked GlcpA. (3) The linkage anal-
ysis data also showed terminal Rhap being the only deoxyHexose
Japan). Methyl b-glycosides of b-(1?3)- and b-(1?6)-linked
D-
galacto-oligosaccharides were synthetic materials described previ-
ously.56,57 dGA (molecular mass of ꢀ10 kDa and DP ꢀ60) was
prepared from a partial acid hydrolysis (0.25 M CF3CO2H for 2 h

78
N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
100
90
80
70
60
50
40
30
20
10
0
5115
0
386.0
729.2
1072.4
Mass (m/z)
1415.6
1758.8
2102.0
Figure 6. MALDI TOF Mass Spectrometry of oligosaccharide products (peracetylated) released from dGA galactanase hydrolysis. Deduced oligosaccharide structures are
presented in Table 4.
Table 4
Oligosaccharide structures deduced from MALDI-TOF MS analysis of peracetylated supernatant (supt) fraction of the Streptomyces sp. exo-b-(13)-D-galactanase hydrolysed dGA
product
Peaks (m/z)
% Intensity (relative intensity)
10
Deduced and possible predominant structures
673.2
(4%)
Hex1HexA1
GlcpA-(1?6)-Galp
Hex2
Galp-(1?6)-Galp
Hex1HexA2
659.1 (701—acetyl group)
701.2
933.2
10
12
12
(10%)
(5%)
GlcpA-(1?4)-GlcpA-(1?6)-Galp
Hex2HexA1
GlcpA-(1?6)-Galp-(1?6)-Galp
919.2 (961—acetyl group)
961.2
977.1 (961.2 K + adduct)
1177.2
30
100
10
(62%)
10
(4%)
Hex2HexA1Pent1
GlcpA-(1?3)-Araf-(1?6)-Galp-(1?6)-Galp
GlcpA-(1?6)-Galp-(1?3)-Araf-(1?6)-Galp
Hex2HexA1DeoxyHex1
Rhap-(1?6)-Galp-(1?4)-GlcpA-(1?6)-Galp
Rhap-(1?4)-GlcpA-(1?6)-Galp-(1?6)-Galp
1149.2 (1191.2—acetyl group)
1191.2
18
15
(15%)
Unless otherwise stated, ions appear as sodium adducts.
at 90–95 °C) of GA (A. senegal, Sigma–Aldrich, St. Louis, Missouri,
USA).43,44 Reactive Black 5 dye (RB5, Sigma–Aldrich, St. Louis,
Missouri, USA) coupled dGA (RB5-dGA) was prepared as described
by Ling et al.43,44
Healthcare Life Sciences, Australia) and a Beckman System Gold
HPLC (Fullerton, USA) at room temperature. The column was
washed with 3 bed vols of binding buffer, and the bound proteins
eluted with a stepwise increase in NaCl to 0.2 M over 100 min,
maintained at 100 mM for 20 min and increased to 1 M over the
next 50 min and maintained at this concentration for 20 min and
then reduced to 0 M over 10 min. Protein elution was monitored
by absorbance at 280 nm. Fractions (2 mL) were collected and
stored at 4 °C for enzyme assays. The fractions with b-galactanase
activity were pooled (EMD Peak 2) and concentrated to 1.8 mL
(7 mg protein) and then buffer exchanged into TSK HW 55 (S) run-
ning buffer (150 mM sodium acetate, pH 5 with 0.2 M NaCl) using
Amicon ultra-15 units (MWCO 10,000; Millipore, Australia). EMD
Peak 2 was chromatographed on a TSK HW 55 (S) column
(1.5 cm ꢃ 83.2 cm, Merck KGaA, Germany) in TSK HW 55 (S) run-
ning buffer at gravitational flow (ꢀ0.06 mL/min). Fractions were
collected every 30 min. The gel filtration column was calibrated
using the following standards from Sigma–Aldrich (St. Louis,
Missouri, USA): albumin from bovine serum (67 kDa), ovalbumin
from chicken egg white (43 kDa), carbonic anhydrase from bovine
erythrocytes (29 kDa) and aprotinin from bovine lung (6.5 kDa).
4.3. Enzyme purification
Purification was conducted at 4 °C unless otherwise indicated.
The column fractions were desalted and buffer exchanged between
chromatography steps on pre-equilibrated EconoPac 10DG (BioRad,
Hercules, USA) disposable columns unless otherwise stated. Protein
in the culture filtrate of the Streptomyces isolate was concentrated
by precipitation with 90% (w/v) ammonium sulfate, followed by
three chromatography steps. The precipitate was dissolved in DEAE
binding buffer (25 mM Tris–HCl, pH 8) and the solution (47 mL,
323 mg protein) loaded under gravitational flow onto a DEAE
Trisacryl (M) column (1.5 cm ꢃ 12.3 cm; PALL BioSepra Life
Sciences, New York, USA) pre-equilibrated in binding buffer. The
unbound protein was collected and the column was washed with
4 bed vols of buffer. The DEAE unbound fraction was concentrated
and the buffer was exchanged into EMD binding buffer (25 mM cit-
ric acid–Na2HPO4, pH 3.5) by ultrafiltration on a YM 10 membrane
(MWCO 10,000; Millipore, Australia).
4.4. Assays
The DEAE unbound fraction (111 mg protein) was applied to a
ꢁ
Fractogel EMD SO3 (M) column (1 cm ꢃ 29.3 cm, Merck KGaA,
b-Galactanase activity was determined using a chromogenic
Germany) pre-equilibrated in EMD binding buffer (25 mM citric
acid–Na2HPO4, pH 3.5) at 1 mL/min using a 50 mL Superloop (GE
assay.44 The assay mixture contained 0.05 mL sample (ꢀ10
lg
protein) and 0.25 mL dyed dGA substrate (RB5-dGA, 0.04 g/mL)

N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
79
prepared in sodium acetate buffer (0.1 M, pH 5). The mixture was
incubated at 37 °C for 20 min, terminated by adding 1.25 mL of
the cold precipitating reagent [20% (v/v) acetic acid, 80% (v/v)
2-methoxyethanol and 0.4% (w/v) zinc acetate] and released
products measured at 590 nm and activity determined as described
in Ling et al. (2009).44
Glycosidase activities were determined using the respective
synthetic p-nitrophenyl (PNP) glycosides as described by
Tsumuraya et al.37 with modification. The assay mixture contained
(50 mM Tris–HCl, pH 7.6; 100 mM NaCl; 50 mM EDTA; 2% SDS;
0.2% PVP; 0.1% b-mercaptoethanol), and extracting with phenol/
chloroform/isoamylalcohol (25:24:1), phenol/chloroform (1:1)
and chloroform. DNA was precipitated from the aqueous phase
with isopropanol, and treated with RNase.
Degenerate primers (Supplementary Table 2) based on SGalase1
peptide sequences were used to amplify internal portions of SGa-
lase1 and SGalase2. Primer pairs were DF1 and DR1 for SGalase1,
and DF2 and DR1 for SGalase2. All PCR products were subcloned
into the pGEM-T Easy vector (Promega) according to the manufac-
turer’s instructions, and sequenced by Macrogen on ABI 3730xl
DNA Analyser machines.
Flanking 5’ and 3’ gene sequence was obtained using the adap-
tor-ligation PCR method of Zheng et al.,61 following digestion of
genomic DNA with restriction enzymes NaeI, NruI, PvuII and SmaI.
The following nested, gene-specific primers (Supplementary Table
2) were used: AF1, AF2 and AF3 for SGalase1, and AR1, AR2, AF4,
AF5, AF6 and AF7 for SGalase2.
the substrate (25
tate buffer (0.1 M, pH 5). The mixture (50
l
L of 3.3 mM) and enzyme, both in sodium ace-
L final volume) was
l
incubated at 37 °C for 15–30 min (or 24 h for the specificity study),
and the reaction terminated with sodium carbonate (200 mM,
0.2 mL) and absorbance was then measured in a 96-well microtitre
plate at 405 nm. One unit (U) of glycosidase activity is defined as
the amount of enzyme that liberates 1 nmol of p-nitrophenol per
minute under the assay conditions described.
Reducing sugar content was measured with the copper arseno-
molybdate reagent of Nelson58 as modified by Somogyi.59 The as-
say was modified to a micro-scale consisting of enzyme and
4.7. Phylogenetic analysis
substrate mixture (50
mixture was incubated for 20 min in a boiling water bath and
cooled on ice before arsenomolybdate reagent (50 L) was added.
The absorbance of the final assay mixture (140 L each) was mea-
lL), and the copper reagent (50 lL). The
The catalytic domain sequences of SGalase 1 and SGalase 2 were
aligned with those of the biochemically characterised GH 43 family
members for phylogenetic analysis using MEGA5.62 Analysis in-
volved 34 amino acid sequences (positions containing gaps and
missing data were eliminated) to construct a phylogenetic tree
using the Neighbor-Joining method.
l
l
sured in a 96-well micro plate at 490 nm. Galactose (Gal) was used
as the standard.
The soluble protein concentration was determined by BioRad
protein assay according to the micro-assay procedure in the sup-
plier’s instruction manual (BioRad, Hercules, USA). Bovine serum
albumin (BSA, Sigma–Aldrich, St. Louis, Missouri, USA, A7030-
50G) was used as the standard.
4.8. Expression and purification of recombinant exo-b-(1?3)-
galactanases
The coding sequences of mature SGalase1 and SGalase2 pro-
teins were amplified by PCR, using gene-specific primers GF1,
GR1, GF2 and GF2 (Table 1). Products were ligated into the
Gateway entry vector pENTR/D-TOPO (Invitrogen) according to
the manufacturer’s instructions, then transferred into pTOOL7,
a Gateway-enabled vector derived from pET-32a(+) (Novagen)
and obtained from Natalie Kibble (ACPFG, Adelaide, Australia).
Constructs were transformed into Origami DE3 E. coli cells
(Novagen) by heat-shock. Selected clones were grown in LB
4.5. Electrophoresis and protein sequencing
The purified enzyme (4
lg protein) was run on a Novex IEF gel
(according to the instructions of the manufacture) and the pI esti-
mated by comparison with the Novex IEF markers pI 3–10 (20
per lane; Invitrogen, Carlsbad, USA).
lL
Aliquots of the fractions (6–8 lg protein) were analysed by
SDS–PAGE on Nu PAGE 4–12% Bis-Tris pre-cast gels (1 mm ꢃ 10
well, Invitrogen, Carlsbad, USA) according to the manufacturer’s
instructions. The gel was stained with GelCode blue stain reagent
(Pierce, Rockford, Illinois, USA) and calibrated with molecular
weight markers (Fermentas Life Sciences, Canada).
Proteins in the SDS–PAGE gel were electro-blotted onto a PVDF-
Plus-membrane (Osmonics Nitrobind 0.45 lm, Minnetonka, USA)
in a BioRad Trans blot cell (Biorad, Hercules, USA). The protein
band with Mr ꢀ45 kDa was excised from the membrane and se-
quenced by Edman N-terminal amino acid sequencing (Biomolec-
ular Research Facility, University of Newcastle, Newcastle,
Australia). Internal peptide sequence was obtained from similar
SDS–PAGE gels that were stained with Coomassie Brilliant Blue
R-250, and de-stained with de-staining reagent (5% acetic acid,
50% methanol). The gel was rinsed in water before the protein
bands (Mr ꢀ45 kDa and ꢀ66.2 kDa) were excised and trypsin in-
gel digested and analysed essentially as described in Natera et
al.60 The amino acid sequences were manually cluster aligned to
the amino acid sequences with the top hit of the BLAST search
medium containing ampicillin (50 lg/mL), induced with
0.5 mM IPTG, and harvested at 3 h post-induction. Inclusion
bodies containing the recombinant enzymes were purified using
BugBuster Master Mix (Novagen) according to the manufac-
turer’s instructions, then solubilised in 8 M urea. Enzymes were
renatured by dialysis against 20 mM sodium acetate buffer, pH
5. A third construct containing YFP was designed and analysed
alongside SGalase 1 and SGalase 2 to ensure that any activity
obtained by the recombinantly expressed protein preparations
was specific to the enzymes and not an endogenous E. coli pro-
tein. The YFP preparation displayed no enzyme activity in any of
the assays used.
4.9. pH and temperature optimum
All buffers were prepared with reference to Dawson et al.63
Glycine–HCl buffer (0.05 M) for pH 2.2, 2.6 and 3, sodium acetate
buffer (0.1 M) for pH 3.8 and 5, MES–NaOH buffer (0.05 M) for
pH 6, MOPS–KOH buffer (0.05 M) for pH 7 and Tris–HCl buffer
(0.05 M) for pH 8. The activity of the enzyme in the different
buffers was determined using the reducing sugar assay.
on the NCBI database, as well as to the exo-1,3-D-galactanase se-
quences made available from the Carbohydrate Active Enzymes
(CAZy) database (http://www.cazy.org/CAZY).
The temperature optimum of the purified enzyme was deter-
mined using dGA as the substrate at the following temperatures:
1 °C, 6 °C, 24 °C, 32 °C, 36.5 °C, 48 °C, 52.5 °C and 63.5 °C in gly-
cine–HCl buffer (pH 3) as described above.
4.6. Cloning of exo-b-(1?3)-galactanase genes
Total genomic DNA was purified by grinding freeze-dried
Streptomyces sp. tissue, homogenising in extraction buffer

80
N. X.-Y. Ling et al. / Carbohydrate Research 352 (2012) 70–81
4.10. Determination of kinetic parameters
Acknowledgements
Michaelis constant (Km) and maximum velocity (Vmax) of the en-
zyme towards dGA at pH 3.8 and 37 °C were determined by a
Lineweaver–Burk plot.64 The substrate concentration was varied
Microbial isolation work for the source of enzyme was carried
out by Kathryn Davis (Department of Microbiology and Immunol-
ogy, University of Melbourne). All sample-derived tryptic peptide
analyses were run by Dr. Siria Natera, and the MS and MS/MS data
were interpreted and de novo sequenced by Ms. Kristina Ford and
Dr. Siria Natera (School of Botany, The University of Melbourne).
This project was supported by funds from the Co-operative Re-
search Centre (CRC) for Bioproducts and Melbourne International
Research Scholarship (MIRS).
over the range of 4.2–83.3 mg/mL, using 0.6 lg protein of the en-
zyme. The initial reaction velocity was determined by measuring
the amount of reducing sugar increase per unit time using the
reducing sugar assay.
4.11. Substrate specificity
Supplementary data
The activity of the enzyme towards various substrates was mea-
sured using the reducing sugar assay. The reaction mixture con-
tained the enzyme at a concentration of 0.51 mg/mL in TSK 55
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2012.02.033.
(S) running buffer (0.05
(1.25 mM for methyl glycosides of b-(1?6)-linked
oligosaccharides, 2.5 mM for methyl glycosides of b-(1?3)-linked
-galacto-oligosaccharides, 1.25 mg/mL for b-(1?3)-galactan and
lg
or
1
lg
protein) and substrate
D-galacto-
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