Journal of Biotechnology 128 (2007) 6171

Induction and characterization of an unusual

-d-galactosidase from Talaromyces avus

a a , Helena Pelantova a ,
Pavla Simerska a , Daniela Monti b, , Ivana Cechov
Martina Mackova c , Karel Bezouska d , Sergio Riva b , Vladimr Kren a,
a

Institute of Microbiology, Academy of Sciences of the Czech Republic, Vdenska 1083, CZ-142 20 Prague 4, Czech Republic
b Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, I 201 31 Milan, Italy
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technicka 5, CZ 168 20 Prague 6, Czech Republic
d Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, CZ-12840 Prague 2, Czech Republic
Received 28 March 2006; received in revised form 23 August 2006; accepted 14 September 2006

Abstract
An extracellular -d-galactosidase from Talaromyces avus CCF 2686 with extremely broad and unusual acceptor specificity is
produced exclusively in the presence of the specific inducer6-deoxy-d-glucose (quinovose). The procedure for the preparation
of this very expensive substance has been modified and optimized. Surprisingly, any of other common -d-galactosidase inducers
or substrates, e.g., d-galactose, melibiose and raffinose, did not stimulate its production. The crude -d-galactosidase preparation
was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The
main isoenzyme (Gal1) was further purified by cation-exchange chromatography and fully characterized. When compared with
other -galactosidases and also with other isoenzymes produced by T. avus, it showed a markedly different regioselectivity
and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum,
guar gum) and significantly inhibited by -d-galactopyranosyl azide, d-galactose, d-xylose, melibiose, methyl - and -dgalactopyranoside and lactose.
2006 Elsevier B.V. All rights reserved.
Keywords: -d-Galactosidase; Talaromyces avus; 6-Deoxy-d-glucose (quinovose); Enzyme purification; Inhibition

1. Introduction
Corresponding author. Tel.: +39 02 2850 0038;
fax: +39 02 2890 1239.
Corresponding author. Tel.: +420 2 9644 2510;
fax: +420 2 9644 2509.
E-mail addresses: daniela.monti@icrm.cnr.it (D. Monti),
kren@biomed.cas.cz (V. Kren).

0168-1656/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.09.006

-d-Galactosidase (-d-galactopyranoside galactohydrolase, E.C. 3.2.1.22) catalyzes the hydrolysis of

simple and complex oligo- and polysaccharides containing terminal -d-galactosyl groups and can be
found in microorganisms (Ulezlo and Zaprometova,

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P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

1982), plants (Chinen et al., 1981) and humans (Beier

et al., 1990). -d-Galactosidases are powerful tools
for oligosaccharide synthesis employing transglycosylation or reverse hydrolysis reactions (Kren and
Thiem, 1997). Several industrial applications of d-galactosidases are known, mainly in sugar industry, where they improve crystallization of sucrose by
hydrolysis of raffinose (Linden, 1982). Moreover, they
can enhance the bleaching effect in pulp and paper
industry (Ratto et al., 1993) and can be used for hydrolysis of raffinose, stachyose and leguminous polysaccharides present in soybean milk (Kotwal et al., 1998).
-d-Galactosidases are also of interest in biomedical
applications, e.g., for treatment of Fabrys disease by
enzyme replacement therapy (Fuller et al., 2004) or
blood type conversion (Olsson et al., 2004).
In our previous study, crude -d-galactosidase
from Talaromyces avus CCF 2686 was shown to
have markedly different regioselectivity in transglycosylation reactions when compared with other fungal enzymes (Weignerova et al., 2001). This enzyme
proved to have extremely broad and unusual substrate
specificity and was able to accept substrates such as 6 O-acetyl (or butyryl)-lactose (Weignerova et al., 1999)
or sterically hindered alcohols, e.g., tert-butyl alcohol
(Simerska et al., 2003, 2006). This alcohol is usually
inert to hydrolases and specifically to glycosidases (Van
Rantwijk et al., 1999) thus, it is often used as a cosolvent in transglycosylation reactions catalyzed by
these enzymes.
Here, we present detailed information on the
production and purification of the T. avus -dgalactosidase, which has an obvious biotechnological
potential. Preparatory synthesis of 6-deoxy-d-glucose
(quinovose) that is essential for production of this
enzyme was also improved and optimized.
2. Materials and methods
2.1. Chemicals and substrates
Unless otherwise stated, all chemicals were of analytical grade and were purchased from SigmaAldrich.
2.2. Analytical methods
Thin-layer chromatography (TLC): precoated silica gel DC-Alufolien Kieselgel 60 F254 plates, Merck.

Flash chromatography: silica gel 60 (4063 m),

Merck. Enzymatic activities were measured using
a spectrophotometer Shimadzu UV-1202 (Japan).
1 H and 13 C NMR spectra were measured on
a VarianUNITY Inova-400 spectrometer (399.87 and
100.55 MHz, respectively) in D2 O or CD3 OD at 30 C.
2.3. Organism and cultivation conditions
T. avus CCF 2686 was obtained from the Culture Collection of Fungi (CCF), Department of Botany,
Charles University, Prague, Czech Republic. Conical flasks (500 ml) with 100 ml medium were inoculated from slants (agar with malt extract, Imuna
sske Michalany, Slovakia) with the suspension
Sari
of spores in 0.1% (v/v) Tween 80. The flasks were
cultivated on a rotary shaker at 28 C. Medium
used [g/l]: yeast extract 0.5, mycological peptone 5,
KH2 PO4 3, NH4 H2 PO4 5, pH 6.0, supplemented with
enzyme inducer 6-deoxy-d-glucose (1 g/l) before sterilization. Different -d-galactosidase inducers or substrates (d-galactose, melibiose, raffinose, stachyose,
methyl -d-galactopyranoside, 6-deoxy-d-galactose
(d-fucose), d-glucose, 2-deoxy-d-glucose, 6-O-tosyld-glucose, 6-chloro-6-deoxy-d-glucose, d-arabinose,
d-xyloseeach 1 g/l) were tested as inducers of this
enzyme using the same conditions as in case of 6deoxy-d-glucose. After the sterilization each flask was
supplemented with sterile 10% (w/v) MgSO4 7 H2 O
solution (5 ml/l).
2.4. Enzyme activity and protein assay
-d-Galactosidase activity was determined
using 2 mM p-nitrophenyl--d-galactopyranoside
(pNPGal) as a substrate. One unit of the -d-galactosidase activity was defined as the amount of enzyme
releasing 1 mol of p-nitrophenol per minute in 50 mM
citratephosphate buffer, pH 4.0 and 30 C. Production
of p-nitrophenol was monitored spectrophotometrically by stopping the 10 min reactions under alkaline
conditions (0.1 M Na2 CO3 , detection at 420 nm), or
continuously at 348 nm (the pH-independent isosbestic
point of the ionized p-nitrophenol). Reactions were
carried out in 1-cm pathlength cells for discontinuous
assays and in 1-mm pathlength cells for continuous assays. The absorption-coefficient difference
at 348 nm between the substrate and the product

P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

was 2698 M1 cm1 in 50 mM citratephosphate

buffer, which is in accordance with literature data
(Selwood and Sinnott, 1988). Protein concentration
was determined according to the method of Bradford
(1976) (Bio-Rad Protein Assay), using bovine serum
albumin as a standard.
2.5. 6-Deoxy-d-glucose-optimized preparatory
procedure
2.5.1. Phenyl 6-O-tosyl--d-glucopyranoside (2)
TsCl (4.82 g, 25.39 mmol) was portion-wise
added at 0 C to a stirred solution of phenyl d-glucopyranoside (1, 5 g, 19.53 mmol) in pyridine
(50 ml). The mixture was incubated at 40 C and
monitored by TLC (CHCl3 :MeOH = 9:1). After 24 h,
another portion of TsCl (2.22 g) was added and
the reaction was terminated after 2 days. The reaction mixture was diluted with AcOEt (50 ml) and
toluene (10 ml) and extracted 10 times with H2 O
(250 ml). The organic phase was washed with 5% (v/v)
HCl (50 ml), H2 O (50 ml), dried over Na2 SO4 and
evaporated. Compound 2 was crystallized from acetone/toluene = 1:2 (4.6 g, white microcrystalline powder, yield 57.5%).
2: 1 H NMR (D2 O, 30 C): 2.212 (3 H, s, CH3 C=), 3.230 (1 H, dd, J3,4 = 9.1 Hz, J4,5 = 9.9 Hz, H4), 3.250 (1 H, dd, J1,2 = 7.8 Hz, J2,3 = 9.2 Hz, H-2),
3.327 (1 H, dd, J2,3 = 9.2 Hz, J3,4 = 9.1 Hz, H-3), 3.515
(1 H, ddd, J4,5 = 9.9 Hz, J5,6d = 2.1 Hz, J5,6u = 5.5 Hz,
H-5), 4.102 (1 H, dd, J5,6u = 5.5 Hz, J6d,6u = 11.5 Hz,
H-6u), 4.244 (1 H, dd, J5,6d = 2.1 Hz, J6d,6u = 11.5 Hz,
H-6d), 4.778 (1 H, d, J1,2 = 7.8 Hz, H-1), 6.785 (2
H, m, ortho-Ph), 6.952 (1 H, m, para-Ph), 7.073 (2
H, AA BB , J = 9.4 Hz, meta-Ts), 7.164 (2 H, m,
meta-Ph), 7.522 (2 H, AA BB , J = 9.4 Hz, orthoTs). 13 C NMR (D2 O, 30 C); HMQC readouts: 20.8
(CH3 -C=), 69.0 (C-4), 69.4 (C-6), 72.8 (C-2), 73.1 (C5), 75.4 (C-3), 116.6 (2 ortho-Ph), 123.5 (para-Ph),
127.9 (2 ortho-Ts), 129.9 (2 meta-Ph), 130.1 (2
meta-Ts). 1 H NMR (CD3 OD, 30 C): 2.369 (3 H, s,
CH3 -C=), 3.311 (1 H, dd, J3,4 = 8.9 Hz, J4,5 = 9.8 Hz,
H-4), 3.393 (1 H, dd, J1,2 = 7.5 Hz, J2,3 = 8.9 Hz, H-2),
3.432 (1 H, dd, J2,3 = 8.9 Hz, J3,4 = 8.9 Hz, H-3), 3.615
(1 H, ddd, J4,5 = 9.8 Hz, J5,6u = 6.2 Hz, J5,6d = 2.0 Hz,
H-5), 4.175 (1 H, dd, J5,6u = 6.2 Hz, J6d,6u = 10.9 Hz,
H-6u), 4.392 (1 H, dd, J5,6d = 2.0 Hz, J6d,6u = 10.9 Hz,
H-6d), 4.829 (1 H, d, J1,2 = 7.5 Hz, H-1), 7.020 (2

63

H, m, ortho-Ph), 7.050 (1 H, m, para-Ph), 7.256 (2

H, m, meta-Ts), 7.290 (2 H, m, meta-Ph), 7.728 (2
H, m, ortho-Ts). 13 C NMR (CD3 OD, 30 C): 21.84
(CH3 -C=), 70.89 (C-6), 71.33 (C-4), 75.02 (C-2), 75.34
(C-5), 78.06 (C-3), 102.30 (C-1), 118.16 (2 orthoPh), 123.81 (para-Ph), 129.36 (2 ortho-Ts), 130.70
(2 meta-Ph), 131.17 (2 meta-Ts), 134.41 (ipso-Ts),
146.67 (para-Ts), 159.23 (ipso-Ph); d, downfield and u,
upfield.
-Glcp: set of vicinal 1 H coupling constants (7.5,
8.9, 8.9 and 9.8 Hz); 1-O-Ph: H-1 is coupled to Cipso of Ph; 6-O-Ts: downfield shift of both H-6s and
C-6.
2.5.2. Phenyl 6-deoxy--d-glucopyranoside (3)
2 (4.6 g, 11.2 mmol) was dissolved in anhydrous THF (80 ml) under argon atmosphere. LiAlH4
(1.52 g, 40 mmol) was added, the reaction mixture was stirred at 0 C until complete dissolution
of the reagents. Then the mixture was refluxed
at 60 C and the reaction course was monitored
by TLC (AcOEt:MeOH:H2 O = 9:0.5:0.5). After 24 h
the solution was slowly poured on ice with water
(250 ml), acidified with 5% (v/v) HCl solution, filtered with Celite, diluted with saturated NaCl solution (50 ml) and extracted with AcOEt (five times,
100 ml). Organic phase was dried with Na2 SO4 , filtered
and evaporated. Crystallization from acetone/toluene
(1:2) and further purification by flash chromatography (CHCl3 :MeOH = 9:1) gave 1.86 g of 3 (yield
69 %).
3: 1 H NMR (D2 O, 30 C): 1.329 (3 H, d,
J5,6 = 6.2 Hz, H-6), 3.112 (1 H, dd, J3,4 = 9.3 Hz,
J4,5 = 9.4 Hz, H-4), 3.452 (1 H, dd, J2,3 = 8.5 Hz,
J3,4 = 9.3 Hz, H-3), 3.470 (1 H, dd, J1,2 = 7.5 Hz,
J2,3 = 8.5 Hz, H-2), 3.496 (1 H, dq, J4,5 = 9.4 Hz,
J5,6 = 6.2 Hz, H-5), 4.901 (1 H, d, J1,2 = 7.5 Hz, H-1),
7.020 (1 H, m, para-Ph), 7.070 (2 H, m, ortho-Ph),
7.295 (2 H, m, meta-Ph). 13 C NMR (D2 O, 30 C): 18.38
(C-6), 73.70 (C-5), 75.49 (C-2), 77.15 (C-4), 78.01
(C-3), 102.42 (C-1), 118.06 (2 ortho-Ph), 123.69
(para-Ph), 130.68 (2 meta-Ph), 159.38 (ipso-Ph); d,
downfield and u, upfield.
A secondary methyl at 1.329 ppm is an evidence
for a 6-deoxy sugar. The phenyl group is present in
the molecule, according to the coupling of H-1 to C-5
it is attached to C-1. The sugar ring is six-membered
(pyranose) since H-1 is coupled to C-5. Set of vicinal

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P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

coupling constants (7.5, 8.5, 9.3 and 9.4) is consistent

with a -gluco configuration providing thus final structure confirmation.
2.5.3. 6-Deoxy-d-glucose (4)
3 (300 mg, 1.25 mmol) was dissolved in 30 ml water
and Dowex 50WX2-100 in H+ cycle (20 mg) was
added. The reaction was refluxed for 24 h, filtrated,
acidified to pH 5.0 by acetic acid and extracted with
diethyl ether (3 10 ml) and then with ethyl acetate
(10 ml) (to remove phenol). The water phase was evaporated, re-dissolved in water and passed through an
Amberlite XAD-2 column (5 cm 8 cm) to remove all
traces of aromatic compounds. Compound 4 was finally
obtained by lyophilization as amorphous solid (172 mg,
84% yield).
4 (-Anomer): 1 H NMR (D2 O, 30 C): 1.019 (3H,
d, J5,6 = 6.3 Hz, H-6), 2.905 (1H, dd, J3,4 = 9.2 Hz,
J4,5 = 9.7 Hz, H-4), 3.301 (1H, dd, J1,2 = 3.8 Hz,
J2,3 = 9.8 Hz, H-2), 3.420 (1H, dd, J2,3 = 9.8 Hz,
J3,4 = 9.2 Hz, H-3), 3.655 (1H, dd, J4,5 = 9.7 Hz,
J5,6 = 6.3 Hz, H-5), 4.936 (1H, d, J1,2 = 3.8 Hz, H-1).
13 C NMR (D O, 30 C): 17.02 (C-6), 67.67 (C-5),
2
72.01 (C-2), 72.77 (C-3), 75.48 (C-4), 92.23 (C-1).
4 (-Anomer): 1 H NMR (D2 O, 30 C): 1.048 (3H,
d, J5,6 = 6.2 Hz, H-6), 2.923 (1H, dd, J3,4 = 9.2 Hz,
J4,5 = 9.5 Hz, H-4), 3.007 (1H, dd, J1,2 = 8.0 Hz,
J2,3 = 9.4 Hz, H-2), 3.196 (1H, dd, J2,3 = 9.4 Hz,
J3,4 = 9.2 Hz, H-3), 3.256 (1H, dd, J4,5 = 9.5 Hz,
J5,6 = 6.2 Hz, H-5), 4.385 (1H, d, J1,2 = 8.0 Hz, H-1).
13 C NMR (D O, 30 C): 17.02 (C-6), 72.17 (C-5),
2
74.66 (C-2), 75.17 (C-4), 75.75 (C-3), 95.99 (C-1); d,
downfield and u, upfield.
The 1 H NMR spectrum contains two sets of signals;
the sample is a mixture of two components in the ratio
1:3. The extracted vicinal constants are consistent with
-Glcp and -Glcp, respectively. Protons H-6 resonate
as a secondary methyl finally proving the structure to
be 6-deoxy-d-glucose.
2.6. Purication of the T. avus -d-galactosidase
2.6.1. Anion-exchange chromatography
Protein suspension obtained by ammonium sulfate precipitation (80% saturation) of a 1 l culture
filtrate (3-day-old culture) from T. avus was centrifuged (20 min at 13,000 rpm and 4 C) and dissolved in 10 mM potassiumphosphate buffer, pH 7.0.

The enzyme solution (42 ml) was dialyzed against

10 mM potassiumphosphate buffer, pH 7.0 (2 5 l)
and after centrifugation (20 min, 13,000 rpm), it was
applied to a Fractogel EMD DEAE-650 (S) (Merck)
column (16 mm 140 mm), equilibrated with 10 mM
potassiumphosphate buffer, pH 7.0, at a flow rate of
1.5 ml/min. After loading, the column was washed with
280 ml of the equilibration buffer and bound proteins
were eluted by a linear gradient from 0 to 0.5 M NaCl
in the same buffer within 3 h at 1.5 ml/min flow rate.
At the end of the gradient, the column was washed
with buffer containing 1 M NaCl until complete protein elution. Fractions (4.5 ml) were assayed for -dgalactosidase activity. Protein concentration was monitored at 280 nm.
2.6.2. Cation-exchange chromatography
The main -d-galactosidase isoenzyme (Gal1)
obtained after DEAE chromatography was dialyzed
(2 5 l) against 5 mM sodium acetate buffer, pH 5.0, the
pH was slowly lowered to pH 4.0 (by 1 M acetic acid)
and, after centrifugation (20 min, 13,000 rpm), it was
applied to a Fractogel EMD SO3 (S) (Merck) column
(16 mm 85 mm), equilibrated with 5 mM sodium
acetate buffer, pH 4.0, at a flow rate of 1.5 ml/min. Proteins were eluted by a linear gradient 00.5 M NaCl in
the same buffer within 3 h at 1.5 ml/min. At the end of
the gradient, the column was washed with buffer containing 1 M NaCl until complete protein elution. Purified proteins were dialyzed against 5 mM ammonium
formate buffer, pH 5.0 (5 l), lyophilized and stored at
20 C.
2.7. Polyacrylamide gel electrophoresis and
zymogram analysis
Enzyme purity was monitored by SDS-PAGE (10%
T, 4% C) according to the method of Laemmli (1970).
The gels were stained with Coomassie Brilliant Blue
and molecular mass under denaturing conditions was
determined by comparison with standard markers (BioRad).
-Galactosidase activity staining (zymogram) was
performed after non-denaturing PAGE (6% T, 4% C) by
incubation of the gel in a solution of 5 mg 1-naphthyl-d-galactose and 5 mg Fast Blue RR Salt in 20 ml of
50 mM citratephosphate buffer, pH 6.0.

P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

2.8. Biochemical characterization of Gal1

N-terminal sequencing (Protein Sequencer
LF3600D, Beckman) was performed according to
the standard protocol provided by the instrument
manufacturer using samples electroblotted onto the
polyvinylidene difluoride (PVDF) membrane. Enzymatic deglycosylation was performed with PNGase
F (peptide-N4 -(N-acetyl--glucosaminyl) asparagine
amidase F, EC 3.5.1.52, New England Biolabs,
UK) following respective protocol provided by the
company.
pH optimum was determined in accordance to the
previously described discontinuous activity assay performed at pH values between 3.0 and 6.0. Under optimal pH (pH 4.0), temperature optimum and stability
were determined using Gal1 (3 mU) and pNPGal at
different temperatures (3070 C) and incubation times
(10 min22 h).
Transglycosylation with tert-butyl alcohol was performed by reaction of tert-butyl alcohol (0.5 ml)
and pNPGal (10 mg, 0.03 mmol) in 50 mM citrate
phosphate buffer, pH 4.0 (0.5 ml). Purified Gal1 (1.6
U, 13 g) was added and the reaction mixture was
shaken at 37 C for 2.5 h. The course of the reaction was
monitored by TLC (AcOEt:MeOH:H2 O = 8:1.5:0.3).
Substrate specificity was evaluated using pNPGal,
4-nitrophenyl and 2-nitrophenyl 2-acetamido-2deoxy--d-galactopyranosides, methyl - and -dgalactopyranosides, -d-galactopyranosyl azide,
lactose, maltose, melibiose, raffinose, stachyose
(2 mM), 50 mM citratephosphate buffer, pH 4.0 and
-d-galactosidase (42 mU/ml). The reactions were
incubated at 30 C for 24 h and analyzed by TLC
(AcOEt:MeOH:H2 O = 7:4:1.5). Substrate specificity
with polymeric substrates guar gum and locust
bean gum was investigated using saturated substrate
solutions (50 mM sodium acetate buffer, pH 4.0) and
-d-galactosidase (80 mU/ml). The reactions were
incubated at 30 C and released d-galactose was determined according to SomogyiNelson method (Paleg,
1959). Kinetic parameters Km and Vmax were determined from MichaelisMenten and LineweaverBurk
plots using SigmaPlot 9.0 (Systat Software GmbH, D).
The reactions were performed using the continuous
activity assay at 30 C and pH 4.0 with pNPGal
(0.024 mM) and the enzyme (12 mU/ml). Inhibition
of the -d-galactosidase was investigated using

65

pNPGal (2 mM) and potential inhibitor (2 mM)

in discontinuous assays. Inhibition constants KI of
-d-galactopyranosyl azide and d-galactose were
determined by continuous enzyme activity assays
using pNPGal (0.52 mM) and inhibitor (0.42 mM).
The same characterizations were performed with
crude enzyme for comparison.

3. Results and discussion

During the search for new glycosidase activities
for the enzymatic synthesis of alkyl glycosides and
oligosaccharide derivatives, the T. avus CCF 2686
strain was shown to produce an -d-galactosidase
activity with very interesting synthetic properties. In
fact, this enzyme was fairly stable over a wide range
of temperatures and in a large number of solvents
(Simerska et al., 2003), and exhibited a novel broad
synthetic potential (Simerska et al., 2006) that makes
it useful for various biotechnological applications. The
aim of this work was, therefore, to identify and characterize the enzymatic activity produced by this fungal
strain for subsequent cloning and over-expression.
3.1. Induction of the -d-galactosidase production
from T. avus
During our previous studies of induction of extracellular glycosidases in several filamentous fungi
(Hunkova et al., 1999), the -d-galactosidase activity
from T. avus CCF 2686 was obtained under induction of quinovose (6-deoxy-d-glucose, 4, Scheme 1).
Although the mechanism of the induction observed
with quinovose was not clear, the effect of this deoxysugar on the stimulation of the production of the
enzyme seemed to be quite specific, as it was not
obtained in the presence of raffinose, a common galactosidase inducer.
During this work, we tested several -d-galactosidase inducers or substrates (d-galactose, melibiose,
raffinose, stachyose, methyl -d-galactopyranoside,
6-deoxy-d-galactose (d-fucose), d-glucose, 2-deoxyd-glucose, 6-O-tosyl-d-glucose, 6-chloro-6-deoxy-dglucose, d-arabinose, d-xylose) as possible inducers
of this enzymatic activity, but none of them gave an
appreciable enzyme production, thus confirming the
exclusive and quite uncommon effect of quinovose.

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P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

Scheme 1. Synthesis of quinovose (6-deoxy-d-glucose).

In the presence of quinovose, maximum volumetric activity of the extracellular -d-galactosidase produced by T. avus was observed after 3 days of
growth (0.24 U/ml, Fig. 1). A correlation between
the release of enzymatic activity and the amount of
quinovose in the culture medium was observed, with
1 g/l of this inducer required for appreciable production
of -d-galactosidase activity, whereas no intracellular -d-galactosidase activity was detected (data not
shown).

Fig. 1. Production of extracellular T. avus -d-galactosidasetime

course of the enzyme activity () and total proteins () in the presence of 0.1% (w/v) quinovose.

3.2. Optimized synthesis of quinovose

Quinovose is a very expensive compound (1 mg
=1
EUR, Sigma) and, therefore, in order to scale-up the
enzyme production for further characterization, we had
to develop and optimize its production. As a starting
point, a synthetic protocol for the preparation of benzyl 6-deoxy--d-glucopyranoside (Danieli et al., 1999)
was chosen. In our synthetic scheme, the phenyl group
replaced the benzyl moiety in order to have a better
leaving group while keeping the possibility of separation of intermediates by extraction instead of by tedious
flash chromatographies. Phenyl -d-glucopyranoside
(1, Scheme 1) was selectively tosylated at the 6-position
and a large variety of conditions (temperature, concentrations, time) were tested for this reaction. It was
found that high temperatures in the tosylation step
could cause decomposition of the product or the production of undesired by-products (di-tosyl and 6-chloro
derivatives). Good quality of TsCl is essential for the
best yields. Subsequent reduction of phenyl 6-O-tosyl-d-glucopyranoside (2) was accomplished by LiAlH4
and the desired product was released by acid hydrolysis catalyzed with a cation-exchanger in H+ form
(final yield 33% related to starting material 1). Cleavage of the phenyl group from phenyl 6-deoxy--dglucopyranoside could be accomplished also enzymatically by different -glucosidases (data not given), but

P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

Fig. 2. Purification of -galactosidases from T. avus (dashed line)

by DEAE chromatography from total culture supernatant proteins
(solid line) in relation to NaCl gradient (dotted line).

acidic hydrolysis was found to be more efficient and

easier. Purification procedures at each step were optimized as well.
3.3. Purication of the T. avus -d-galactosidase
In order to obtain a suitable amount of the T.
avus -d-galactosidase for subsequent characterization, enzyme production was scaled-up to 1 l of culture
and proteins released into the culture medium were collected after 3 days of growth.
Purification by anion-exchange chromatography
revealed three different -d-galactosidase isoenzymes
in the culture filtrate of T. avus (Fig. 2; Table 1, lines
35). The major activity, Gal1 (172 U), was recovered
during the washing step with the equilibration buffer.
It was not adsorbed to the resin, but its elution was
retarded and it could thus be separated from the other
unbound proteins. Gal2 (20 U) was eluted at 0.22 M
NaCl and Gal3 (2.5 U) at 0.18 M NaCl. As shown in

67

Fig. 3. SDS-PAGE (a) and native PAGE (b) analyses of purified T.

avus -d-galactosidases. (M), molecular mass markers (mass in
thousands); (1) crude enzyme; (2) Gal1; (3) Gal2; (4) Gal3 (all
three after cation-exchange chromatography as in Fig. 2); (5) Gal1
stained by Coomassie Blue; (6) Gal1 after zymogram analysis.

Table 1, this purification step was especially effective

in the improvement of Gal1 specific activity. Gal2
and Gal3 were not significantly purified, as also subsequently confirmed by SDS-PAGE analysis (Fig. 3a,
lanes 3 and 4, respectively).
Gal1 was further purified on a cation-exchange
Fractogel SO3 column. After this chromatographic
step, the specific activity of purified Gal1 was
619 U/mg, which corresponded to a 108-fold increase
of specific activity and to a 35% purification yield
(Table 1, line 6).
3.4. Biochemical characterization of Gal1
The purified isoenzyme Gal1 gave a single band of
approximately 63 kDa by SDS-PAGE analysis (Fig. 3a,
lane 2). This molecular mass is similar to those of
other reported fungal -d-galactosidases (Puchart et
al., 2000). Zymogram analysis showed one band on

Table 1
Purification of extracellular -d-galactosidases from Talaromyces avus
Purification step

Total protein [mg]

Activity [U]

Specific activity [U/mg]

Yield [%]

Purification factor

Culture broth (940 ml)

Dialyzed 80% (NH4 )2 SO4 ppt.
Gal1 after Fractogel DEAE
Gal2 after Fractogel DEAE
Gal3 after Fractogel DEAE
Gal1 after Fractogel SO3

40
18.2
0.4
2.2
0.7
0.13

230
195
172
20
2.5
80.5

5.75
10.7
431
9
3.5
619

100
85
75
9
1
35

1
1.8
75
1.6
0.6
108

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P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

Fig. 4. Alignment of N-terminal sequence (N-term) of T. avus Gal1 with sequences of -galactosidases from various sources. Sequence
homology was searched in the NCBI database using the BLASTP tool and alignment for optimal sequence similarity was performed using
the program ClustalX. Species names and NCBI Accession numbers: Z. mrakii, Zygosaccharomyces mrakii (BAA99555); S. paradoxus, Saccharomyces paradoxus (CAA64759); S. cerevisiae, Saccharomyces cerevisiae (AAL07760); S. mikatae, Saccharomyces mikatae (CAA64760);
Z. cidri, Zygosaccharomyces cidri (AAA35280); C. josui, Clostridium josui (BAB83765); P. chrysosporium, Phanerochaete chrysosporium
(AAG24511); U. vinacea, Umbelopsis vinacea (AAB35252); T. delbrueckii, Torulaspora delbrueckii (BAA86883). All sequences aligned with
the N-terminal sequence of Gal1 are representing enzyme precursors before posttranslational modifications.

activity-stained non-denaturing PAGE, which corresponded to the main protein band stained by Coomassie
Blue (Fig. 3b).
N-terminal sequence analysis of Gal1, determined
by the Edman protocol, allowed us to unequivocally
identify the first 20 amino acids as LNNGLAVTPQMGWDDWNAFG. A significant homology of this
sequence of Gal1 to similar sequences of other
-galactosidases was shown by sequence homology
search in the NCBI database using the BLASTP tool.
Alignment for the best sequence similarity, performed
using the program ClustalX, confirmed the identity of
the 63 kDa protein band with Gal1 (Fig. 4). Following
PNGase F endoglycosidase digestion (to remove any
N-linked carbohydrates), no significant mobility shift
of this protein band on SDS-PAGE gel was observed
(data not shown) indicating that Gal1 is not at all or
only scarcely N-glycosylated.
Purified Gal1 was able to catalyze efficiently the
transglycosylation of tert-butyl alcohol in a rather
unique reaction, proving virtually the same behavior of
the crude enzyme (Simerska et al., 2003, 2006) (Fig. 5).
On the contrary, the other two isoenzymes, Gal2 and
Gal3, showed no activity with this alcohol.
Moreover, Gal1 (and plausibly also Gal3) differs
from Gal2, and most of other -d-galactosidases by
the lack of activity towards melibiose (Gal--1,6-Glc)
(Fig. 6), which is a quite common substrate for this
group of glycosidases (Post and Luebke, 2005; King et
al., 2002).

Therefore, the hydrolytic activity towards melibiose

observed when using the crude enzymatic preparation
is specifically related to the presence of the isoenzyme Gal2. Surprisingly, Gal1 was found to cleave
d-galactose from raffinose and stachyose, the -1,6galactosylated derivatives of sucrose, indicating that
this enzyme can recognize bulky substrates as well.
That was indeed proved with galactomannans such
as guar gum and locust bean gum (Fig. 7) as, e.g.,
in the case of -d-galactosidases from Oryza sativa
(Kim et al., 2002) or from Thermomyces lanuginosus
(Puchart et al., 2000). -Galactosyl azide was scarcely
hydrolyzed by Gal1, whereas pNPGalNAc and galactosides were not substrates.
Gal1 showed optimal activity at pH 3.54.5
(Fig. 8a), which is quite typical for fungal -d-galactosidases (Ozsoy and Berkkan, 2003), and 50 C
(Fig. 8b). Moreover, the enzyme remained fully
stable for 22 h at temperatures up to 40 C and was
inactivated only after 5 h at temperatures above 60 C.
A good thermostability of -d-galactosidase from T.
avus might be advantageous in its further biocatalytical applications. Km and Vmax measured with
pNPGal at 30 C were calculated to be 0.54 0.07
mM and 0.21 0.01 mmol/min. Inhibition of -dgalactosidase activity was observed in the presence
of 2 mM -d-galactopyranosyl azide (75%), dgalactose (51%), d-xylose (49%), melibiose (46%),
methyl -d-galactopyranoside (15%), methyl -dgalactopyranoside (13%) and lactose (12%). In similar

P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

69

Fig. 5. Synthesis of tert-butyl--d-galactopyranoside catalyzed by purified T. avus Gal1. For experimental details see Section 2.

experimental conditions, no remarkable effect was

observed with 2-acetamido-2-deoxy-d-galactopyranose, d-galactosamine hydrochloride, d-glucose,
2-deoxy-d-glucose, 6-deoxy-d-glucose, 2-acetamido-

2-deoxy-d-glucopyranose, d-glucosamine hydrochloride, saccharose, stachyose, d-fructose and d-arabinose. Inhibition constants of -d-galactopyranosyl
azide and of d-galactose for -d-galactosidase were

Fig. 6. Reaction scheme and thin layer chromatography of the hydrolysis of melibiose by -d-galactosidase from T. avus crude enzyme (1),
Gal1 (2), Gal2 (3) and Gal3 (4) isoenzymes.

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P. Simerska et al. / Journal of Biotechnology 128 (2007) 6171

Fig. 7. Degalactosylation of polymeric substrates (locust bean gum:

 and guar gum: ) catalyzed by T. avus Gal1. The reactions
were performed with Gal1 (80 mU/ml) at saturated concentrations
of substrates and 30 C. Released d-galactose was expressed as a
percentage of d-galactose present originally in the galactomannan,
assuming d-galactose/d-mannose ratio is 23:77 and 38:62 for locust
bean gum and guar gum, respectively.

determined to be KI = 0.25 0.05 and 0.38 0.03 mM,

respectively.
In conclusion, production and purification of -dgalactosidase from T. avus were studied in detail and
a preparatory procedure of its inducer was described.
The identification and characterization of the main d-galactosidase present in the crude preparation allows
undertaking our future work for cloning and sequencing of the entire gene coding for the enzyme. By homology modeling with known -galactosidase structures,
our studies aim to provide structural understanding of
the rather unique synthetic properties of T. avus -dgalactosidase.

Acknowledgements
This work was supported by Czech Science Foundation (grant 203/05/0172), Czech Ministry of Education (OC D25.002 and LC06010), EU project COST
D25/0001/02 and NATO (Collaborative project No.
LST.CLG.980125 to S.R. and V.K.).

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