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 resulting filtrate h
Trang 1family 35 b-galactosidase of Hypocrea jecorina with
Christian Gamauf1, Martina Marchetti2, Jarno Kallio3, Terhi Puranen3, Jari Vehmaanpera¨3,
Gu¨nter Allmaier2, Christian P Kubicek1and Bernhard Seiboth1
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
(http://www.cazy.org/) [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: gamauf@mail.zserv.tuwien.ac.at
Website: http://www.vt.tuwien.ac.at/
(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 aryl-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.
Trang 2cellulolytic 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 (http://www.expasy.org/tools/ 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 60C for 1 h also led to recovery of almost all of the original activity, but temperatures over 65C 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
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
Trang 3ble- 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 assumpglycosyla-tion 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-liumsp 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 60C, and
a substantial decrease was noted at alkaline pH and at temperatures > 60C 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 (kcat⁄ Km) 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 Km, as well as a higher
Vmax 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
*
*
*
100
200
300
[FU]
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.
Trang 4Although the activity was only slightly reduced in the
presence of 10 mm d-glucose (data not shown),
d-galactose had a significant impact on oNPG
hydro-lysis (Fig 5) The inhibition was competitive with an
inhibition constant Kiof 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].
Trang 5HPLC 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 (kcat) 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 kcat⁄ Km 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 kcat⁄ Km, 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 correscorres-ponding sugar acid (lactobionic acid) severely impaired the catalytic efficacy This was in higher proportions due to a decrease in kcat, 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, lacd-galac-tose 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
pH
0
20
40
60
80
100
10 20 30 40 50 60 70 80
Temperature [°C]
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 (m M ) V max (nkatÆmg)1) k cat (s)1) k cat ⁄ K m (LÆg)1Æs)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
Trang 6The 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 Km and Vmax for lactose : pNPG, as
deter-mined in this study, in consideration, an actual lactose
hydrolysing activity of 0.4 lmolÆ(minÆg mycelial dry
weight))1for 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))1or 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 Km 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, 18 mm; Aspergillus oryzae, 36–180 mm; A niger, 54–99 mm; Kluyveromyces
fragil-is, 15–52 mm; K lactfragil-is, 35 mm) [18,19] In addition, the ratio of Ki to Km 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 Ki,Gal⁄ Km,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
Galactose [mM]
0.25 mM oNPG 0.75 m M oN PG
A
0
0.02
0.04
0.06
0.08
0.10
0.12
-2
0.25 m M oNPG 0.75 m M oNPG
Galactose [m M]
Ki
B
0.25 m M 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 Ki.
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]
0
0
2
2
4
4
6
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 m M (B) lactose by Bga1 Peaks: 1, transglycosylation product; 2, lactose; 3, glucose; 4, galactose.
Trang 7Species 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, (NH4)2SO42.8, KH2PO44, MgSO4Æ7H2O 0.6, CaCl20.6, FeSO4Æ7H2O 0.005, MnSO4ÆH2O 0.0016, ZnSO4Æ7H2O 0.0014, CoCl20.002 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 28C and the pH between 4.8 and 5.2 by addition of 12.5% (w⁄ v) NH4OH
or 17.5% (w⁄ v) H3PO4, 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, 4C) and aliquots of the culture supernatant were stored at )80 C until use The supernatant was then centrifuged at 15 000 g and 4C 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 lL) 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)1Æs)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
Trang 8(final concentration in the basic assay was 3 mm) to give a
total reaction volume of 1 mL The assay was incubated at
30C for 30–60 min and stopped by the addition of 3 mL
of 1 m Na2CO3 Absorbance was measured at 405 nm
(eoNP¼ 4530 LÆmol)1Æcm)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 30C The reaction
was stopped by boiling the incubation mixture at 95C 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 H2SO4 as the
mobile phase at a flow rate of 0.5 mLÆmin)1 (35C) 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 lL 10 mm
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 30C)
The amount of NADH (eNADH¼ 6300 LÆmol)1Æcm)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 Kmand Vmax, 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 Km and
Vmax values The errors represent the standard deviation
from the measurement and regression The molecular mass
of Bga1 used for the calculation of kcatwas 118 789 Da
Temperature and pH optimum and enzyme
stability
To determine the optimal temperature and pH for the assay
described above, different temperatures (15–75C) and
0.1 m McIlvaine buffer (citric acid⁄ Na2HPO4pH 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 (TOF2, Shimadzu Biotech, Manchester, UK) equipped with a nitrogen laser (k¼ 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)
Trang 9[31] Extracted tryptic peptides were desalted and step-wise
purified utilizing ZipTip technology [32] (C18 reversed
phase, standard bed, Millipore, Bedford, MA) by loading
the extracted peptide mixture onto C18-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)1in water) as well as the dried-droplet
tech-nique for 2,5-dihydroxybenzoic acid (10 mgÆmL)1in water)
Positive-ion mass spectra were recorded on the same
instru-ment as instru-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)
References
1 Coutinho PM & Henrissat B (1999)
Carbohydrate-active enzymes: an integrated database approach In
Recent Advances in Carbohydrate Bioengineering(Gilbert
HJ, Davies G, Henrissat B & Svensson B, eds), pp
3–12 Royal Society of Chemistry, Cambridge
2 Holsinger VH & Kligerman AE (1991) Application of
lactase in dairy foods and other foods containing
lac-tose Food Technol 45, 92–95
3 Gekas V & Lo´pez-Leiva M (1985) Hydrolysis of lactose:
a literature review Process Biochem 20, 2–12
4 Rings EH, Grand RJ & Buller HA (1994) Lactose
intol-erance and lactase deficiency in children Curr Opin
Pediatr 6, 562–567
5 Nakayama T & Amachi T (1999) b-Galactosidase,
enzy-mology In Encyclopedia of Bioprocess Technology:
Fer-mentation, Biocatalysis and Bioseparation(Flickinger
MC & Drew SW, eds), pp 1291–1305 Wiley, New
York, NY
6 Boon MA, Janssen AE & van’t Riet K (2000) Effect of
temperature and enzyme origin on the enzymatic
syn-thesis of oligosaccharides Enzyme Microb Technol 26, 271–281
7 Penttila¨ M, Limon C & Nevalainen H (2004) Molecular biology of Trichoderma and biotechnological applica-tions In Handbook of Fungal Biotechnology (Arora DK, ed.), pp 413–427 Marcel Dekker, New York, NY
8 Seiboth B, Hartl L, Salovuori N, Lanthaler K, Robson
GD, Vehmaanpera¨ J, Penttila¨ ME & Kubicek CP (2005) Role of the bga1-encoded extracellular b-galac-tosidase of Hypocrea jecorina in cellulase induction by lactose Appl Environ Microbiol 71, 851–857
9 Harman GE, Howell CR, Viterbo A, Chet I & Lorito
M (2004) Trichoderma species – opportunistic, avirulent plant symbionts Nat Rev Microbiol 2, 43–56
10 Salovuori I, Makarow M, Rauvala H, Knowles J & Ka¨a¨ria¨inen L (1987) Low molecular weight high-mannose type glycans in a secreted protein of the filamentous fun-gus Trichoderma reesei Bio⁄ Technology 5, 152–156
11 Rojas AL, Nagem RA, Neustroev KN, Arand M, Adamska M, Eneyskaya EV, Kulminskaya AA, Garratt
RC, Golubev AM & Polikarpov I (2004) Crystal struc-tures of b-galactosidase from Penicillium sp and its complex with galactose J Mol Biol 343, 1281–1292
12 Hung MN & Lee BH (2002) Purification and characteri-zation of a recombinant b-galactosidase with transgalac-tosylation activity from Bifidobacterium infantis HL96 Appl Microbiol Biotechnol 58, 439–445
13 Mayer J, Conrad J, Klaiber I, Lutz-Wahl S, Beifuss U
& Fischer L (2004) Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lac-tulose J Agric Food Chem 52, 6983–6990
14 Willfo¨r S, Sjo¨holm R, Laine C & Holmbom B (2002) Structural features of water-soluble arabinogalactans from Norway spruce and Scots pine heartwood Wood Sci Technol 36, 101–110
15 Voragen AGJ, Pilnik W, Thibault JF & Renard CMGC (1995) Pectins Food Polysaccharides and Their Applica-tions(Stephen AM, ed.), pp 287–339 Marcel Dekker, New York, NY
16 Crueger A & Crueger W (1984) Carbohydrates Biotech-nology(Kieslich K, ed.), pp 421–457 Verlag Chemie, Weinheim
17 Sinnott ML (1990) Catalytic mechanisms of enzymic glycosyl transfer Chem Rev 90, 1171–1202
18 Aehle W (2004) Enzymes in Industry Wiley-VCH, Wein-heim
19 Jurado E, Camacho F, Luzo´n G & Vicaria JM (2002)
A new kinetic model proposed for enzymatic hydrolysis
of lactose by a b-galactosidase from Kluyveromyces fra-gilis Enzyme Microb Technol 31, 300–309
20 Onishi N & Tanaka T (1995) Purification and properties
of a novel thermostable galacto-oligosaccharide-produ-cing b-galactosidase from Sterigmatomyces elviae CBS8119 Appl Environ Microbiol 61, 4026–4030
Trang 1021 Yanahira S, Suguri T, Yakabe T, Ikeuchi Y, Hanagata
G & Deya E (1992) Formation of oligosaccharides from
lactitol by Aspergillus oryzae b-d-galactosidase
Carbo-hydr Res 232, 151–159
22 Gopal PK, Prasad J & Gill HS (2003) Effects of the
consumption of Bifidobacterium lactis HN019
(DR10TM) and galacto-oligosaccharides on the
micro-flora of the gastrointestinal tract in human subjects
Nutr Res 23, 1313–1328
23 Charalampopoulos D, Wang R, Pandiella SS & Webb
C (2002) Application of cereals and cereal components
in functional foods: a review Int J Food Microbiol 79,
131–141
24 Benitez T, Rincon AM, Limon MC & Codon AC
(2004) Biocontrol mechanisms of Trichoderma strains
Int Microbiol 7, 249–260
25 Knee EM, Gong FC, Gao M, Teplitski M, Jones AR,
Foxworthy A, Mort AJ & Bauer WD (2001) Root
mucilage from pea and its utilization by rhizosphere
bacteria as a sole carbon source Mol Plant Microbe
Interact 14, 775–784
26 Seiboth B, Hartl L, Pail M, Fekete E, Karaffa L &
Kubicek CP (2004) The galactokinase of Hypocrea
jecorinais essential for cellulase induction by lactose but
dispensable for growth on d-galactose Mol Microbiol
51, 1015–1025
27 Seiboth B, Hofmann G & Kubicek CP (2002) Lactose metabolism and cellulase production in Hypocrea jecor-ina: the gal7 gene, encoding galactose-1-phosphate uri-dylyltransferase, is essential for growth on galactose but not for cellulase induction Mol Genet Genom 267, 124– 132
28 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA & Stuhl K (2006) Current Protocols in Molecular Biology Greene⁄ Wiley Inter-science, New York, NY
29 Mueller O, Hahnenberger K, Dittmann M, Yee H, Dubrow R, Nagle R & Ilsley D (2000) A microfluidic system for high-speed reproducible DNA sizing and quantitation Electrophoresis 21, 128–134
30 Kussmann M, Nordhoff E, Rahbek-Nielsen H, Haebel
S, Rossel-Larsen M, Jakobsen L, Gobom J, Mirgorods-kaya E, Kroll-Kristensen A, Palm L et al (1997) Mat-rix-assisted laser desorption⁄ ionization mass
spectrometry sample preparation techniques designed for various peptide and protein analytes J Mass Spec-trom 32, 593–601
31 Shevchenko A, Wilm M, Vorm O & Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels Anal Chem 68, 850–858
32 Pluskal MG (2000) Microscale sample preparation Nat Biotechnol 18, 104–105