Báo cáo khoa học: Characterization of the bga1-encoded glycoside hydrolase family 35 b-galactosidase of Hypocrea jecorina with galacto-b-D-galactanase activity pdf

Characterization of the bga1-encoded glycoside hydrolase
family 35 b-galactosidase of Hypocrea jecorina with
galacto-b-
D-galactanase activity
Christian Gamauf
1
, Martina Marchetti
2
, Jarno Kallio
3
, Terhi Puranen
3
, Jari Vehmaanpera
¨
3
,
Gu
¨
nter Allmaier
2
, Christian P. Kubicek
1
and Bernhard Seiboth
1
1 Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Austria
2 Research Area Instrumental Analytical Chemistry, Institute of Chemical Technology and Analytics, Vienna University of Technology, Austria
3 Roal Oy, Rajama
¨
ki, Finland
The enzyme b-galactosidase (EC 3.2.1.23) catalyses the
hydrolysis of terminal nonreducing b-d-galactose

residues in b-d-galactosides as, for example, lactose
(1,4-O-b-d-galactopyranosyl-d-glucose) and structur-
ally related compounds. It is found in plants and
animals, as well as in a wide variety of microorganisms
including yeasts, fungi, bacteria and Archaea. Accord-
ing to the Carbohydrate Active Enzymes database
( [1], b-galactosidases are
members of four different glycoside hydrolase (GH)
families (GH1, GH2, GH35 and GH42), indicating
their structural diversity. In biotechnology, they are
mainly applied for the hydrolysis of lactose to
d-glucose and d-galactose in various products of the
dairy industry [2,3]. This results in improved quality of
the end product (softer texture of ice cream, faster
ripening of cheese, etc.) and also lessens the problem
of lactose intolerance, which is prevalent in more than
half of the world’s population [4]. In addition, b-gal-
actosidases catalyse transgalactosylation reactions of
various b-d-galactosides including lactose. Recently,
these galacto-oligosaccharides have attracted consider-
able interest because of a proposed beneficial effects
on human health [5,6].
The filamentous fungus Hypocrea jecorina (ana-
morph: Trichoderma reesei) is a potent producer of
Keywords
b-galactosidase; galactanase;
Hypocrea jecorina; substrate specificity;
transglycosylation
Correspondence
C. Gamauf; Research Area Gene

Technology and Applied Biochemistry,
Institute of Chemical Engineering, Vienna
University of Technology, Getreidemarkt
9 ⁄ 166-5, A-1060 Vienna, Austria
Fax: +43 158 801 17299
Tel: +43 158 801 17265
E-mail:
Website: />(Received 16 January 2007, accepted 22
January 2007)
doi:10.1111/j.1742-4658.2007.05714.x
The extracellular bga1-encoded b-galactosidase of Hypocrea jecorina
(Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1)
promoter region and purified to apparent homogeneity. The monomeric
enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa
(MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several
disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl-
and alkyl-b-d-galactosides. Based on the catalytic efficiencies, lactitol and
lactobionic acid are the poorest substrates and o-nitrophenyl-b-d-galacto-
side and lactulose are the best. The pH optimum for the hydrolysis of gal-
actosides is  5.0, and the optimum temperature was found to be 60 °C.
Bga1 is also capable of releasing d-galactose from b-galactans and is thus
actually a galacto- b- d-galactanase. b-Galactosidase is inhibited by its reac-
tion product d-galactose and the enzyme also shows a significant trans-
ferase activity which results in the formation of galacto-oligosaccharides.
Abbreviations
ACN, acetonitrile; GH, glycoside hydrolase; LIF, laser-induced fluorescence; oNPG, o-nitrophenyl-b-
D-galactopyranoside; pNPG, p-nitrophenyl-
b-
D-galactopyranoside.
FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS 1691

cellulolytic and hemicellulolytic enzymes and due to its
strong promoters and excellent secretion capacities it is
also of interest for the expression of heterologous
proteins [7]. One of the carbon sources used for cellulase
production is lactose, which requires an extracellular
b-galactosidase activity for being assimilated by the
fungus. The gene encoding this enzyme, bga1, has
recently been described [8]. Like most other fungal
b-galactosidases, the encoded protein belongs to GH35.
The ability of filamentous fungi to assimilate and
grow on lactose is enigmatic, as lactose is unlikely to
occur in their natural environment. This argument is
also substantiated by the finding that bga1 is induced
by d-galactose and l-arabinose, thus pointing to a role
for the enzyme in the degradation of plant poly- and
oligosaccharides which are present in the natural hab-
itat of a rhizosphere-competent and potentially endo-
phytic fungus like Trichoderma [9]. Consequently, our
aim was to purify Bga1 and study its substrate profile.
We reasoned that this information may provide us
with a hint towards the role of this enzyme in the
physiology of the fungus, as well as to its potential use
in biotechnology.
Results
Purification of b-galactosidase Bga1
In order to facilitate the purification of the bga1-enco-
ded b-galactosidase, we fused its ORF to the promoter
region of the pyruvate kinase-encoding pki1 gene and
cultivated the resulting recombinant strain on
d-glucose. Under these conditions, b-galactosidase

formation parallels growth, and the culture superna-
tant was thus harvested when two-thirds of the carbon
source had been consumed. The resulting filtrate had a
specific activity of 83.5 nkatÆmg
)1
(with o-nitrophenyl-
b-d-galactopyranoside; oNPG) and analysis by
SDS ⁄ PAGE showed a protein band in the expected
molecular mass range (110 kDa; Fig. 1), which already
accounted for a significant part of the total secreted
protein. Concentration by ultrafiltration and subse-
quent purification by gel filtration and cation-exchange
chromatography (Table 1) yielded a single protein
band at  110 kDa, proving a homogenously purified
protein. The specific activity with oNPG was
828.2 nkatÆmg
)1
, indicating a roughly tenfold enrich-
ment over the original activity.
Physicochemical properties and stability of Bga1
The size of denatured H. jecorina Bga1, measured
using SDS ⁄ PAGE (see above), is  110 kDa. Because
virtually the same molecular mass was determined by
gel-permeation chromatography of the native enzyme,
we conclude that the enzyme is a monomer. The iso-
electric point of the unfolded protein was determined
as 6.6, which is in good agreement with the value
calculated from the amino acid sequence (6.35) using
the protparam tool ( />protparam.html), indicating the absence of any post-
translational modification altering the charge of the

protein (e.g. phosphate, sulfate).
Bga1 is stable over a broad range of pH values,
which extends far into the alkaline pH range and inac-
tivation occurs rapidly only below pH 3.0 (data not
shown). Incubation of the enzyme at different tempera-
tures up to 60 °C for 1 h also led to recovery of almost
all of the original activity, but temperatures over 65 °C
led to rapid denaturation (data not shown).
Bga1 is a glycoprotein
MALDI-TOF-MS in the linear mode was used to
determine the exact molecular mass, and a value of
118 789 ± 485 Da was obtained based on single-, dou-
1
kD
200
150
120
85
100
70
60
50
40
23
4
Fig. 1. SDS ⁄ PAGE of the purified Bga1. Lanes: 1, molecular size
marker; 2, culture supernatant; 3, combined factions after gel filtra-
tion; 4, purified protein after ion-exchange chromatography. Each
lane contained 10 lg protein.
Table 1. Purification of the H. jecorina b-galactosidase.

Step
Total
protein
(mg)
Total
activity
(nkat)
Specific
activity
(nkatÆmg
)1
)
Yield
(%)
Enrichment
(fold)
Culture filtrate 24.45 2041.2 83.5 100.0 1
Gel filtration 1.29 693.0 535.8 34.0 6.4
Ion-exchange
chromatography
0.32 261.0 828.2 12.8 9.9
b-Galactosidase of Hypocrea jecorina C. Gamauf et al.
1692 FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS
ble- and triple-charged molecular ions (Fig. 2A). This
is higher than the theoretical value calculated from
the amino acid sequence of the mature protein
(109 301 Da). The asymmetric peak of the singly
charged molecule indicates a heterogeneity which was
also observed in the double- and triple-charged mole-
cules at the high mass side of the peaks (indicated by

asterisks in Fig. 2A). Capillary gel electrophoresis-
on-the-chip confirmed that the isolated protein exhibits
heterogeneities which are again reflected as an asym-
metric peak shape (Fig. 2B, tailing is marked with an
asterisk).
Because Bga1 is an extracellular protein, glycosyla-
tion was expected, and this assumption was supported
by glycoprotein staining (data not shown). Taking the
average size of Trichoderma N-glycosylation structures
of 1500 Da into account [10], the difference between
the theoretical and the determined molecular mass of
9500 Da (see above) would predict the presence of
six N-glycosylation antennae. Analysis of the Bga1
amino acid sequence using the NetNGlyc tool (http://
www.cbs.dtu.dk/services/NetNGlyc/) revealed the pres-
ence of 11 consensus sites for N-linked glycosylation at
positions 287, 402, 434, 536, 544, 627, 709, 782, 810,
836 and 930. Because Bga1 is orthologous to a Penicil-
lium sp. b-galactosidase, for which the 3D structure
and the attached N-glycans have been determined [11],
we compared the positions in the two proteins. This
analysis (Fig. 3) showed that of the seven positions
identified in Penicillium sp., four are conserved in
Bga1. All these positions are located at the surface of
the jelly roll domain of Bga1 and thus most probably
are glycosylated in vivo. To further substantiate these
findings, in gel digestion of the highly purified protein
was carried out. Seventeen peptides resulting from
Bga1, evenly distributed over the protein, could be
detected by MALDI-RTOF-MS (Fig. 3). This sums up

to a sequence coverage of 12.4% for the mature pro-
tein (without the predicted signal peptide). The detec-
ted peptides represent amino acid sequences not
bearing potential N-glycosylation sites. This failure in
detecting glycopeptides despite step-wise elution to
overcome suppressing effects during the MALDI pro-
cess might be explained by the much higher ionization
efficiency for nonglycosylated peptides which were
preferentially eluted in the solution containing 50 and
75% (v ⁄ v) acetonitrile (ACN) where also most of the
N-glycosylated peptides were expected. None of these
tryptic peptides contained a consensus for potential
N-glycosylation, and thus none of the sites mentioned
above could be falsified.
Optimal temperature and pH for catalysis
The optimal temperature and pH for the reaction of
Bga1 with oNPG as substrate was determined (Fig. 4):
maximal activity was found at pH 5.0 and 60 °C, and
a substantial decrease was noted at alkaline pH and at
temperatures > 60 °C. The enzyme retained more
activity at acidic pH.
Substrate specificity for b-galactosides and
kinetic properties of Bga1
The substrate specificity of the purified H. jecorina
Bga1 was determined towards oNPG, methyl-b-
d-galactoside and various disaccharides, and the kin-
etic constants were calculated (Table 2). The catalytic
efficiencies (k
cat
⁄ K

m
) indicate that oNPG and lactu-
lose (4-O-b-d-galactopyranosyl-d-fructose) are the
best substrates for Bga1. This is the result of both a
lower Michaelis constant K
m
, as well as a higher
V
max
for these substrates. In addition, Bga1 also
showed significant activity with galactobiose (4-O-b-
d-galactopyranosyl-d-galactose), while its activity
with lactitol (4-O-b-d-galactopyranosyl-d-glucitol),
lactobionic acid (4-O-b-d-galactopyranosyl-d-gluconic
acid) and methyl-b-d-galactoside was poor.
The effect of hydrolysis products (each at 10 mm
final concentration) on the hydrolysis of oNPG by
Bga1 under standard assay conditions was studied.
0
50
100
%Int
50 000
100 000
m /z
[M]
+
[M]
3+
[M]

2+
*
*
*
100
200
300
[FU]
20 30 40 50 60
70
[s]
150 000
A
B
Fig. 2. Establishment of the exact molecular mass by MS and pro-
tein purity by capillary gel electrophoresis-on-the-chip. (A) Determin-
ation of the exact molecular mass of H. jecorina Bga1 by positive
ion MALDI-TOF-MS in the linear mode. The singly, doubly and triply
charged ions are indicated by respective symbols. Asterisks mark
the asymmetric peak flank, indicating a heterogenic glycosylation
pattern. (B) Capillary gel electrophoresis-on-the-chip electrophero-
gram of 0.45 lg Bga1 detected by LIF (FU, fluorescence units).
The asterisk again marks the asymmetric peak flank, indicating a
heterogenic glycosylation pattern.
C. Gamauf et al. b-Galactosidase of Hypocrea jecorina
FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS 1693
Although the activity was only slightly reduced in the
presence of 10 mmd-glucose (data not shown),
d-galactose had a significant impact on oNPG hydro-
lysis (Fig. 5). The inhibition was competitive with an

inhibition constant K
i
of 1 mm.
When the reaction products were monitored using
HPLC, formation of transglycosylation products
became evident (Fig. 6). Peaks with different retention
times than the substrates and hydrolysis products of
the reaction were detected with all disaccharides tested,
and the concentration of the putative transglycosyla-
tion products was inversely correlated with that of
d-galactose. The degree of transglycosylation was
dependent on the concentration of the substrate, as
also found in other studies [12,13].
Activity of Bga1 on polymeric substrates
The ability of Bga1 to hydrolyse b-galactosidic bonds
in polymeric substrates was tested with two
b-1,4-galactans (from lupin and potato) and one
b-1,3- ⁄ b-1,6-arabinogalactan (from larch wood).
Fig. 3. Alignment of the b-galactosidases
from H. jecorina and Penicillium sp. Shaded
residues are conserved in both enzymes.
The solid lines indicate the tryptic peptides
detected by MALDI peptide mass finger-
printing. Predicted (H. jecorina; CAD70669)
and confirmed (Penicillium sp.; CAF32457)
N-glycosylation sites are marked by darker
shading and sites conserved in both
sequences by arrows. Diamonds indicate
amino acid residues involved in substrate
binding and catalysis [11].

b-Galactosidase of Hypocrea jecorina C. Gamauf et al.
1694 FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS
HPLC analysis of the reaction products confirmed
that the enzyme released d-galactose in a time- and
concentration-dependent manner. The reaction fol-
lowed a Michaelis–Menten kinetic, and the constants
for the polymeric substrates are given in Table 3.
Turnover numbers (k
cat
) on all three polymeric sub-
strates were in the same range as on galactobiose
(5.68 s
)1
) and significantly higher than on most other
galactosides tested (Table 2). The k
cat
⁄ K
m
ratios are
less favourable, but the lower values can be explained
taking into account the fraction of d-galactose units
in the polymers that are actually accessible for the
b-galactosidase (those that are on the nonreducing
ends of the chains, e.g. 36.8% in arabinogalactan
from larch wood) [14]. The enzyme showed highest
activity against the d-galactose-richest galactan from
lupin, which is consistent with an activity only
against b-galactosidic bounds. This was also
supported by the finding that no other monosaccharides
were detectable during HPLC analysis of the enzymatic

assays. Bga1 was also unable to hydrolyse p-nitro-
phenyl-b-l-arabinofuranoside, which is the most typical
l-arabinose-linkage found in arabinogalactans [15].
Discussion
In this study, we characterized a b-galactosidase from
H. jecorina belonging to GH family 35. Although
b-galactosidases have been purified and characterized
from a variety of sources [3,5,16], their GH family affi-
liation is unknown in most cases, which makes com-
parison of the obtained results difficult. To the best of
our knowledge, this is the first report of the enzymo-
logical properties of a b-galactosidase identified as a
member of GH35.
H. jecorina Bga1 hydrolysed all b-galactosides tes-
ted, but with clearly different preference: based on
the ratio of k
cat
⁄ K
m
, the aromatic artificial galactoside
oNPG was the most preferred substrate, which may
indicate a beneficial role of hydrophobicity in one of
the steps of the hydrolysis. Among naturally occur-
ring galactosides, galactobiose was the best substrate.
Substitution of the O-4-linked hexose by a corres-
ponding polyol (lactitol) or a corresponding sugar
acid (lactobionic acid) severely impaired the catalytic
efficacy. This was in higher proportions due to a
decrease in k
cat

, suggesting that such substitutions
may interfere with proton assistance of the acid⁄ base
residue or the nucleophilic attack on C-1 of d-galac-
tose [17]. Interestingly, lactose was also a comparably
poor substrate for Bga1, which coincides with the low
rate of its assimilation by H. jecorina, and supports
the assumption that lactose is not the natural sub-
strate for Bga1.
0
20
40
60
80
100
23456789
pH
enzyme activity [arbitary units]
0
20
40
60
80
100
10 20 30 40 50 60 70 80
Temperature [°C]
en
z
ym
ea
c

t
ivity [a
rb
ita
r
y
u
nits]
A
B
Fig. 4. pH and temperature optimum of the H. jecorina Bga1. The
activity was assayed at the indicated pH (A) and temperatures (B)
as described in the Experimental procedures.
Table 2. Kinetic parameters of H. jecorina b-galactosidase with various b-galactosides.
Substrate K
m
(mM) V
max
(nkatÆmg
)1
) k
cat
(s
)1
) k
cat
⁄ K
m
(LÆg
)1

Æs
)1
)
o-Nitrophenyl-b-
D-galactopyranoside 0.36 ± 0.01 144.61 ± 0.15 17.31 ± 0.02 159.41 ± 5.41
Galactobiose 9.06 ± 2.48 47.46 ± 6.26 5.68 ± 0.69 1.83 ± 0.51
Lactobionic Acid 15.31 ± 2.34 0.83 ± 0.04 0.10 ± 0.004 0.018 ± 0.003
Lactitol 19.77 ± 2.00 1.04 ± 0.05 0.13 ± 0.01 0.018 ± 0.002
Lactose 8.79 ± 1.00 5.78 ± 0.16 0.69 ± 0.02 0.23 ± 0.02
Lactulose 0.56 ± 0.06 12.74 ± 0.34 1.52 ± 0.04 7.98 ± 0.78
Methyl-b-
D-galactopyranoside 2.85 ± 0.39 0.71 ± 0.03 0.09 ± 0.003 0.15 ± 0.02
C. Gamauf et al. b-Galactosidase of Hypocrea jecorina
FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS 1695
The low affinity for lactose also explains the relat-
ively poor growth of H. jecorina on this carbon source:
Seiboth et al. [8], using p-nitrophenyl-b-d-galacto-
pyranoside (pNPG) as a substrate, reported extra-
cellular Bga1 activity during growth on lactose to be
86 lmolÆ(minÆg mycelial dry weight)
)1
. Taking the
ratio of K
m
and V
max
for lactose : pNPG, as deter-
mined in this study, in consideration, an actual lactose
hydrolysing activity of 0.4 lmolÆ(minÆg mycelial dry
weight)

)1
for lactose can be calculated. Thus, assuming
that H. jecorina grows on an initial lactose concentra-
tion of 10 or 20 gÆL
)1
(80–85% substrate saturation),
this is equivalent to a hydrolysis rate of  20 mg lac-
toseÆ(hÆg mycelial dry weight)
)1
or 0.5 gÆ(dayÆg mycelial
dry weight)
)1
.
Despite being less efficient on lactose than on other
b-galactosides, Bga1 may still offer some advantages
for lactose hydrolysis from a biotechnological per-
spective: although its K
m
for lactose is 8.8 mm, this is
significantly lower than the corresponding values of
other fungal b-galactosidases used commercially (e.g.
Talaromyces thermophilus,18mm; Aspergillus oryzae,
36–180 mm; A. niger, 54–99 mm; Kluyveromyces fragil-
is, 15–52 mm; K. lactis,35mm) [18,19]. In addition,
the ratio of K
i
to K
m
calculated for d-galactose and
lactose, which can be interpreted as a specificity con-

stant that determines preferential binding of the sub-
strate vs. that of the monosaccharide end products, is
0.11, which compares favourably with the K
i,Gal
⁄ K
m,Lac
ratio reported, e.g. for A. oryzae and A. niger (0.01
and 0.006, respectively) [19]. In view of these advan-
tages, the ability of Bga1 to form transglycosylation
products, albeit a known property of GH35 b-galacto-
sidases [5,12,20,21], is also noteworthy. b-Linked
oligosaccharides derived from d-galactose and other
hexoses are of considerable interest, partially as poten-
tial prebiotics for the food industry, as well as in phar-
macology and medicine [22,23].
The main reason for studying the enzymological
properties of Bga1 in more detail was our interest in
the physiological role of this protein in H. jecorina.
0
20
40
60
80
100
0246810
Galactose [mM]
enzyme activity [arbitary units]
0.25 mM oNPG
0.75 mM oN PG
A

0
0.02
0.04
0.06
0.08
0.10
0.12
-2
-4 2 4 6 8 10 12
0.25 mM oNPG
0.75 m
M oNPG
Galactose [mM]
K
i
1/
Var
b
t
a
ry uni
[i t
s
]
B
0.25 mM oNPG
0.75 m
M oNPG
Fig. 5. Inhibition of oNPG hydrolysis by D-galactose. (A) Addition of
D-galactose to the basic b-galactosidase assay (see Experimental

procedures) results in a significant reduction of the enzyme activity.
The reduction can be partially overcome by increasing the substrate
concentration, indicating a competitive inhibition mechanism. (B)
Dixon diagram for determination of the inhibition constant K
i
.
6
6
7
7
4
4
8
8
9
9
10
10
11
11
12
12
13
13
14
14
retention time [min]
retention time [min]
detector units
detector units

0
0
2
2
4
4
6
6
1
1
2
2
3
3
A
B
Fig. 6. Formation of transglycosylation products during lactose
hydrolysis by H. jecorina b-galactosidase. Chromatogram of a partial
hydrolysis of 10 m
M (A) and 100 mM (B) lactose by Bga1. Peaks: 1,
transglycosylation product; 2, lactose; 3, glucose; 4, galactose.
b-Galactosidase of Hypocrea jecorina C. Gamauf et al.
1696 FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS
Species of Trichoderma are known to be able to colon-
ize and grow in the rhizosphere of plants [9,24]. In
bacteria, rhizosphere competence is related to the abil-
ity to utilize arabinogalactan from the root mucilage
[25], and it is likely that similar mechanisms may have
evolved in rhizosphere competent fungi too. In any
case, Bga1 is able to act on polymeric b-1,3- and

b-1,4-galactans. A role for Bga1 in arabinogalactan
degradation may also explain why bga1 expression and
that of the Leloir pathway genes gal1 (encoding galac-
tokinase) and gal7 (encoding galactose-1-phosphate
uridylyltransferase) is induced by both d-galactose and
l-arabinose [26,27]. The ability to attack galactose poly-
mers may also lend to speculate about the structure of
the Bga1 protein, but further studies are necessary to
find out which domains could be involved in recogni-
tion and⁄ or binding to the polysaccharides. Also, it
will be intriguing to learn whether other enzymes act
synergistically with Bga1. A prerequisite for such stud-
ies, however, is a more detailed knowledge of the
structure of the commercially available b-galactans
which can be used as a model for such studies.
Experimental procedures
Substrates
Unless indicated otherwise, all substrates and peptides and
proteins for MS calibration were purchased from Sigma
(St Louis, MO) and at least of analytical grade. Arabino-
galactan was purchased from Fluka (Buchs, Switzerland)
and galactan from lupin and potato was from Megazyme
(Bray, Ireland).
Strain and culture conditions
To purify the extracellular b-galactosidase, H. jecorina strain
PKI-BGA13, a recombinant of QM9414 (ATCC 26921),
which carries multiple copies of a pki1:bga1 cassette allowing
overexpression of Bga1 during growth on d-glucose, was
used.
Construction of the expression vector and of the bga1-

overexpressing strain was performed as described previously
[8]. The fungus was cultivated in a Braun Biostat ED bio-
reactor (working volume 10 L) for 30 h using the following
medium (gÆL
)1
): d-glucose 40, bacto peptone 4, yeast
extract 1, (NH
4
)
2
SO
4
2.8, KH
2
PO
4
4, MgSO
4
Æ7H
2
O 0.6,
CaCl
2
0.6, FeSO
4
Æ7H
2
O 0.005, MnSO
4
ÆH

2
O 0.0016,
ZnSO
4
Æ7H
2
O 0.0014, CoCl
2
0.002. Struktol (1 mLÆL
)1
) was
added at the beginning of the cultivation as an antifoam
agent. The fermenter was inoculated with 200 mL of a
shake flask preculture grown for 65 h in the same medium.
The temperature was kept constant at 28 °C and the pH
between 4.8 and 5.2 by addition of 12.5% (w ⁄ v) NH
4
OH
or 17.5% (w ⁄ v) H
3
PO
4
, respectively. Aeration was 1 vvm,
and Impeller speed was set to 200 r.p.m., and after 12 h
linearly increased to 750 r.p.m. at a rate of 30 r.p.m.Æh
)1
.
Purification of b-galactosidase
The fermenter broth was withdrawn, the biomass separated
by centrifugation (3500 g, 15 min, 4 °C) and aliquots of the

culture supernatant were stored at )80 °C until use. The
supernatant was then centrifuged at 15 000 g and 4 °C for
30 min to remove particulate matter and filtered through
Amicon Ultra columns (Millipore, Bedford, MA) with a
cut-off value of 5000 Da. The concentrated protein solution
( 250 l L) was diluted in 50 mm citrate buffer, pH 5.5
containing 150 mm NaCl, concentrated again (final volume
 500 lL) and loaded onto a HR 16 ⁄ 50 column packed
with Superose 12 prep grade (GE Bioscience, Chalfont,
UK) and equilibrated with the same buffer. Fractions were
collected and assayed for b-galactosidase activity, and those
that contained > 20% of the peak fraction activity were
pooled and concentrated as above. For further purification,
the concentrate was diluted in 10 mm citrate buffer pH 5.5
(buffer A) to a final volume of 10 mL, and loaded onto a
Mono S HR 5 ⁄ 5 column (GE Bioscience) previously equili-
brated with 10 column volumes of buffer A. The column
was then washed with further 10 column volumes of buf-
fer A, and thereafter the bound proteins were eluted by
applying a linear gradient of 0–0.6 mm NaCl in a total of
40 mL of buffer A. Fractions were assayed for b-galactosi-
dase activity and those containing > 20% of the activity in
the peak fraction were pooled.
b-Galactosidase activity assay
Unless stated otherwise, b-galactosidase was assayed by
measuring the hydrolysis of oNPG in 50 mm acetate buffer
pH 5. The reaction was started by the addition of oNPG
Table 3. Kinetic parameters of H. jecorina b-galactosidase with various galactans
Substrate Bound % Gal
K

m
(gÆL
)1
)
V
max
(nkatÆmg
)1
)
k
cat
(s
)1
)
k
cat
⁄ K
m
(LÆg
)1
Æs
)1
)
Arabinogalactan b-1,3 ⁄ b-1,6 79 13.59 ± 1.87 36.50 ± 4.88 4.34 ± 0.58 0.32 ± 0.06
Galactan (Lupin) b-1,4 91 38.34 ± 6.87 57.80 ± 10.32 6.87 ± 1.23 0.18 ± 0.05
Galactan (Potato) b-1,4 87 25.70 ± 4.82 27.93 ± 5.20 3.32 ± 0.62 0.13 ± 0.03
C. Gamauf et al. b-Galactosidase of Hypocrea jecorina
FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS 1697
(final concentration in the basic assay was 3 mm) to give a
total reaction volume of 1 mL. The assay was incubated at

30 °C for 30–60 min and stopped by the addition of 3 mL
of 1 m Na
2
CO
3
. Absorbance was measured at 405 nm
(e
oNP
¼ 4530 LÆmol
)1
Æcm
)1
) against a blank sample. Activ-
ities are given in nanokatals, one nkat being equivalent to
the release of 1 nmol o-nitrophenol per second under the
conditions given above. Specific activities are related to
1 mg of protein, determined by the Bio-Rad Protein Assay
(Bio-Rad, Hercules, CA).
To determine the activity of b-galactosidase on other
substrates, the following procedure was used: 1 lg purified
b-galactosidase was incubated with appropriate amounts of
potential substrates in 50 mm acetate buffer pH 5.0 in a
total volume of 1 mL for 2–24 h at 30 °C. The reaction
was stopped by boiling the incubation mixture at 95 °C for
10 min, and, after cooling on ice and centrifugation in an
Eppendorf centrifuge (5 min), the amount of liberated
d-galactose was determined by HPLC using an Aminex
HPX-87H column (Bio-Rad) with 10 mm H
2
SO

4
as the
mobile phase at a flow rate of 0.5 mLÆmin
)1
(35 °C). The
concentration of d-galactose in the sample was calculated
from a calibration curve and used to determine the nmol of
d-galactose per second formed in the assay.
The activity of b-galactosidase with lactulose could not
be measured in this way, because d-galactose and d-fruc-
tose displayed similar retention times in our HPLC analy-
sis. Therefore, we used d-galactose dehydrogenase (Sigma)
to quantify the produced d-galactose. To this end, the pH
of the incubation mixture (processed as above) was adjusted
to pH 8.6 by addition of 1 m NaOH, then 10 lL10mm
NAD
+
and 0.1 m phosphate buffer pH 8.6 up to 1 mL
were added. The reaction was started by addition of 60 mU
of d-galactose dehydrogenase (incubation: 1 h at 30 °C).
The amount of NADH (e
NADH
¼ 6300 LÆmol
)1
Æcm
)1
) pro-
duced was determined by measuring the absorbance at
340 nm against a blank (incubation mixture without sub-
strate). In these assays one nkat was defined as 1 nmol

d-galactose formed per second under the conditions given
and again related to the protein concentration.
To determine K
m
and V
max
, the activity of Bga1 with the
given substrates was assayed at at least five different sub-
strate concentrations in an appropriate range. Each meas-
urement was performed in triplicate and the Enzyme
Kinetics Module in sigma plot 2001 (Systat Software Inc.,
Point Richmond, CA) was used to calculate the K
m
and
V
max
values. The errors represent the standard deviation
from the measurement and regression. The molecular mass
of Bga1 used for the calculation of k
cat
was 118 789 Da.
Temperature and pH optimum and enzyme
stability
To determine the optimal temperature and pH for the assay
described above, different temperatures (15–75 °C) and
0.1 m McIlvaine buffer (citric acid ⁄ Na
2
HPO
4
pH 3–8) were

used. The stability was investigated by incubating the
enzyme for 1 h at the given pH or temperature and then
assaying the activity as described above. Throughout these
experiments, the enzyme concentration was 1 lgÆmL
)1
.
Biochemical analytical methods
Standard methods, as described previously [28] were used
for SDS ⁄ PAGE, isoelectric focusing and Coomassie Brilli-
ant Blue staining. Glycoprotein staining of SDS ⁄ PAGE gels
was performed with the Pro-Q Emerald 300 Glycoprotein
Gel and Blot Staining Kit (Molecular Probes, Eugene,
OR).
Capillary gel electrophoresis-on-the-chip
Chip-based separation of proteins was performed using a
prototype instrument (2100 bioanalyser) of Agilent Tech-
nologies (Waldbronn, Germany), which has been described
in detail elsewhere [29]. Briefly, this instrument uses lab-on-
a-chip technology to separate high molecular mass proteins
electrophoretically in a linear polymer solution and is cou-
pled to a laser-induced fluorescence (LIF) detector using a
fluorescence dye.
Determination of the molecular mass
The native molecular mass of the b-galactosidase was deter-
mined by gel-permeation chromatography using a 16 ⁄ 70
column filled with Bio-Gel A-1.5 m (Bio-Rad), equilibrated
with 0.1 m acetate buffer pH 5 containing 0.5 m NaCl at a
flow rate of 0.5 mLÆmin
)1
. Bio-Rad Gel Filtration Standard

proteins were used to calibrate the column.
Mass spectrometric characterization
Sample preparation for MALDI-TOF-MS of the native
protein was carried out on a stainless steel target, applying
the dried droplet preparation technique [30] using 2,4,6-
trihydroxyacetophenone (20 mgÆmL
)1
in methanol) as the
matrix. Positive-ion mass spectra were recorded on a
vacuum MALDI-TOF ⁄ curved field reflector TOF instru-
ment (TOF
2
, Shimadzu Biotech, Manchester, UK)
equipped with a nitrogen laser (k ¼ 337 nm) in the linear
mode by accumulating 200–500 single unselected laser
shots. External calibration was performed using an aqueous
solution of b-galactosidase from Escherichia coli and the
mass spectra were treated with the company-supplied
smoothing algorithm.
For in-gel digestion the respective protein band of an
appropriate SDS ⁄ PAGE was excised manually with a stain-
less steel scalpel and digested with trypsin (bovine pancreas,
modified; sequencing grade, Roche, Mannheim, Germany)
b-Galactosidase of Hypocrea jecorina C. Gamauf et al.
1698 FEBS Journal 274 (2007) 1691–1700 ª 2007 The Authors Journal compilation ª 2007 FEBS
[31]. Extracted tryptic peptides were desalted and step-wise
purified utilizing ZipTip
Ò
technology [32] (C
18

reversed
phase, standard bed, Millipore, Bedford, MA) by loading
the extracted peptide mixture onto C
18
-ZipTips which were
activated by ACN ⁄ ultra pure water (1 : 1, v ⁄ v) and further
equilibrated with water. After binding of the sample the
tips were washed two times with 10 lL water for salt
removal. Step-wise elution was performed consecutively
using solutions consisting of 2, 10, 50 and 75% (v ⁄ v) ACN
in water. The different fractions (3 lL each) were analysed
by MALDI-curved field reflectron (RTOF)-MS. Sample
preparation was again carried out on a stainless steel target,
applying the thin-layer preparation technique with sinapic
acid (6 mgÆmL
)1
in water) as well as the dried-droplet tech-
nique for 2,5-dihydroxybenzoic acid (10 mgÆmL
)1
in water).
Positive-ion mass spectra were recorded on the same instru-
ment as mentioned above. External calibration was per-
formed using an aqueous solution of standard peptides
(Bradykinin fragment 1–7, human angiotensin II, somato-
statin and ACTH fragment 18–39).
Acknowledgements
We thank Verena Seidl for her help with the isoelectric
focusing analysis, Roland Mu
¨
ller for his help with

CGE-on-the-chip experiment and Agilent Technologies
for the loan of the instrument. Sanna Hiljanen-Berg
and Sirpa Okko are thanked for skilful technical assist-
ance. Work in the laboratory of CG, CPK and BS was
supported by the Austrian Science Foundation FWF
(P16143).
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