Enzyme and Microbial Technology 40 (2007) 969–975

Production of thermostable ␤-mannosidase by a strain of

Thermoascus aurantiacus: Isolation, partial purification
and characterization of the enzyme
Joseph Gomes  , Katherine Terler, Regina Kratzer,
Elke Kainz, Walter Steiner ∗
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, Graz A-8010, Austria
Received 3 June 2005; received in revised form 28 July 2006; accepted 4 August 2006

Abstract
The production of an extracellular thermostable ␤-mannosidase by the thermophilic fungus Thermoascus aurantiacus Miehe was investigated
in shake flask cultures. Among five different carbon sources and inducers tested, locust bean gum at 15 g l−1 induced the highest (4 nkat ml−1 )
␤-mannosidase activity. Although the fungus grew very well on mannose at an initial concentration of 20 g l−1 there was practically no enzyme
synthesis. Low level of enzyme synthesis occurred only when mannose had dropped to about 1 g l−1 . The ␤-mannosidase was purified to apparent
homogeneity by ethanol precipitation, differential solubility at pH 4.0 and cation ion exchange chromatography. The enzyme had a molecular mass
of 99.9 kDa and a pI value of 4.8. The ␤-mannosidase showed maximum activity at pH 2.5–3.0 and 76 ◦ C. It exhibited highest pH stability at 5.9
after 5 h incubation at 50 ◦ C. It had a half-life of 10 min at 76 ◦ C. The Km and Vmax values for p-nitrophenyl-␤-d-mannopyranoside (p-NP-␤-MP)
were 1.1 mM and 61 nkat mg−1 , respectively. Low transglycosylation activity was observed when the enzyme was incubated with p-NP-␤-MP as
glycosyl donor and methyl-␣-d-mannopyranoside as acceptor at 50 ◦ C and pH 5.0 and small amounts of ␤-configured methylmannobioside was
formed.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Thermoascus aurantiacus; ␤-Mannosidase; Production; Purification; Characterization

1. Introduction ear ␤-(1 → 4)-linked backbone of d-mannose and d-glucose

␤-Mannan-based polysaccharides like mannans, galac-
tomannans, galactoglucomannans and glucomannans are impor-
tant components of higher plant cell walls [1,2]. Mannan is
made of ␤-1,4-linked mannose residues only, while glucoman-
nan has a backbone composed of ␤-1,4-linked glucose and
mannose sugars [1,2]. When the backbone of both mannan
and glucomannan is substituted with ␣-1,6-linked galactosyl
residues these polysaccharides are called galactomannan and
galactoglucomannan, respectively [1,2]. Characteristic features
of galactoglucomannans, predominant in softwood, are a lin-
∗
Corresponding author. Tel.: +43 316 873 8420; fax: +43 316 873 8434.
E-mail address: walter.steiner@TUGraz.at (W. Steiner).
 Unfortunately, Dr. Joseph Gomes passed away on the 20th of March during
the reviewing procedure of this paper. The co-authors gratefully acknowledge
his enthusiasm and support.
residues and O-acetyl groups [1,2]. Galactomannan, function-
ing as a reserve polysaccharide, occurs in large quantities in
leguminous seeds, e.g., locust bean and guar [3] whereas glu-
comannan plays a key structural role in the plant cell walls
of hardwood [2]. The complete hydrolysis of mannan-based
hetero-polysaccharides is achieved by the concerted action of
several enzymes. For example, endo-acting ␤-mannanases (EC
3.2.1.78) catalyze random hydrolysis of ␤-1,4-linkage within the
main chain of ␤-1,4-mannans to short linear or branched ␤-1,4-
mannooligomers, which are further cleaved by the exo-acting
␤-mannosidase (EC 3.2.1.25) to mannose [4,5]). Additional
enzymes like ␣-galactosidase (EC 3.2.1.22), ␤-glucosidase (EC
3.2.1.21) and esterase (3.1.1.72) are required to split off side
residues like galactose, glucose and acetyl group, respectively
[4,5].
Although ␤-mannosidases have been reported to occur in
many bacteria, yeasts, fungi, marine algae, germinating seeds,
invertebrates and vertebrates there are few reports on the purifi-

0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2006.08.011
970 J. Gomes et al. / Enzyme and Microbial Technology 40 (2007) 969–975
cation and characterization of microbial ␤-mannosidases. So
far, purification and characterization of ␤-mannosidases have
been described from fungi, Aspergillus niger [6–8], Aspergillus
awamori [9,10], Sclerotium rolfsii [11], Trichoderma reesei [12],
yeast, Aureobasidium pullulans [13] and bacteria, Cellulomonas
fimi [14], Thermobifida fusca [15], Pyrococcus furiosus [16] and
Thermotoga neapolitana [17]. In addition to its role in the uti-
lization of hemicelluloses by microorganisms, ␤-mannosidase
plays an important role in the lysosomal degradation process
of N-glycosylprotein glycans in higher animals and ruminants
[18]. Genetic disorders associated with the deficiency of lyso-
somal ␤-mannosidase enzyme result in the buildup of the dis-
accharide Man␤-1-4 GlcNAc and the disease ␤-mannosidosis,
which is manifested by mental retardation, skeletal abnormal-
ities, hearing loss and renal failure [18]. Together with ␤-
mannanases, ␤-mannosidases are widely used in the sacchar-
ification of hemicellulose for further conversion to chemicals
and fuels, in the treatment of coffee beans, in the produc-
tion of Konjac, in the hydrolysis of galactomannans used in
oil and gas drilling and in the biobleaching of pulp and paper
[5,19–21]. The enzyme also provides a useful tool for structural
studies of stock polysaccharides having ␤-mannosidic linkages
and for the sequencing of hetero-polysaccharides and carbohy-
drate moieties of numerous glycoproteins [20,21]. In addition
to the biological significance of ␤-mannosidase, there is great
interest in using ␤-mannosidase in the synthesis of oligosac-
charides that have potential as prebiotics and anti-adhesives
[22,23].
The thermophilic fungus Thermoascus aurantiacus has the
capability to degrade a wide variety of cellulosic and lignocel-
lulosic substrates and produce relatively high levels of xylanase
[24–26], endoglucanase, ␤-glucosidase [25] together with low
levels of exocellulase [24,26] and mannanase [26,27]. The cel-
lulolytic and hemicellulolytic enzymes of this fungus are highly
thermostable over a wide range of pH and temperatures and
have tremendous industrial application potential [21,25–28].
Previous reports that T. aurantiacus produce ␤-mannosidase
at very low level (1–3 nkat ml−1 ) when grown on wheat straw
[25] or Solka Floc [26] and evidence of transglycosylation
activity of these crude culture filtrates in the presence of ␤-d-
mannopyranosyl fluoride as donor and methanol and monosac-
charides as acceptors [29] encouraged the authors to continue
their work on T. aurantiacus ␤-mannosidase. The objectives of
the present work were to increase the ␤-mannosidase produc-
tion by selecting the proper inducing carbon source, to purify
and characterize the enzyme and to demonstrate the ability of
the purified enzyme to synthesize manno-oligosaccharides by
transglycosylation.
2. Materials and methods
2.1. Microorganism and culture conditions
T. aurantiacus Miehe, isolated by Dr. I. Gomes at Bangladesh Jute Research
Institute, Dhaka, identified by Centraalbureau voor Schimmelcultures, Baarn,
The Netherlands and deposited under the stock number BT 2079 at the
Culture Collection, Institute of Biotechnology and Biochemical Engineering,
Graz University of Technology, Austria, was used in this study. For main-
tenance, the fungus was grown on potato dextrose agar (PDA) at 47 ◦ C for
4 days and stored at 4 ◦ C for further use. It was subcultured every 3–4
weeks.
2.2. Shake culture experiments
Shake cultures were performed using the basal mineral medium optimized
for xylanase [25], endoglucanase and ␤-glucosidase production [26]. The basal
medium was supplemented with proper carbon and nitrogen sources. The initial
pH of the medium was adjusted to 4.9 prior to the addition of the carbon and
organic nitrogen sources. Shake cultures were performed in 300-ml Erlenmeyer
flasks containing 100 ml culture medium. Each flask medium was inoculated
with a PDA piece (ca. 1 cm2 ) containing an actively growing 4-day-old culture
of T. aurantiacus and incubated at 47 ◦ C and 150 rpm on an orbital shaker for
10–11 days. The culture broth was centrifuged (19,000 × g, 4 ◦ C, 30 min) and
the clear supernatant was used for enzyme and protein assays and for the enzyme
purification process.
2.3. Purification of the β-mannosidase
␤-Mannosidase from the fungus T. aurantiacus, grown on LGB and soymeal,
was purified to apparent homogeneity in three simple steps: ethanol precipitation,
differential solubility at pH 4.0 and cation-exchange chromatography. Proteins in
the crude culture filtrate (320 ml) were precipitated by the dropwise (7 ml min−1 )
addition of cold (4 ◦ C) ethanol (480 ml) under slow stirring. The precipitated pro-
teins containing ␤-mannanase and ␤-mannosidase activities were harvested by
centrifugation, resuspended in 47 ml sodium-formate buffer (25 mM, pH 4.0)
and incubated on ice for 60 min with gentle shaking to dissolve the proteins.
Proteins insoluble at pH 4.0 were collected by centrifugation and then dissolved
at pH 5.0 in sodium citrate buffer (25 mM). The supernatant containing the pH
4.0 soluble proteins showed mainly ␤-mannosidase activity whereas proteins
soluble at pH 5.0 showed mainly ␤-mannanase, but no ␤-mannosidase activity.
One-millilitre portions of protein solution in sodium formate buffer (pH 4.0)
were then loaded on a Mono S® HR 5/5 1 ml cation-exchange column (Pharma-
cia) equilibrated with sodium formate buffer (50 mM, pH 4.0) containing 1 M
NaCl. The column was washed with 5 ml of the same buffer and the proteins
were eluted with a two-step gradient (0 → 1 M NaCl, total volume 16 ml) at a
flow rate of 1 ml min−1 . The first step was set at 0.4 M NaCl after 5 ml of eluent.
This concentration was kept constant for 4 ml of elution volume. In the second
step the concentration of 1 M NaCl was reached after 7 ml of eluent. Fractions
of 1 ml were collected and assayed for ␤-mannosidase activity. ␤-Mannosidase
was eluted in the 11th fraction. Chromatography on Mono S® and gel filtration
on Superdex 200 were performed at room temperature using a FPLC® System
(Pharmacia).
2.4. PAGE electrophoresis
SDS-PAGE was performed on PhastGelTM gradient 8–25 gels from Phar-
macia Biosciences. The proteins were stained with Coomassie brilliant blue.
Native polyacrylamide gel electrophoresis was carried out on 7.5% polyacry-
lamide slab gels in Tris–glycine buffer system at pH 8.3 and 4 ◦ C consistent with
Laemmli’s method [30]. The gels were visualized by staining the protein bands
with 4 mg ml−1 p-nitrophenyl-␤-d-mannopyranoside (p-NP-␤-MP) dissolved in
25 mM sodium citrate buffer (pH 5.0).
2.5. Determination of isoelectric point
The isoelectric point of the enzyme was determined by isoelectrofocusing on
an IEF ready gel (Bio-Rad), IEF pH 3–10, against IEF markers with pI 4.45–9.6
(Bio-Rad), according to the manufacturer’s instructions.
2.6. Determination of molecular mass
The molecular weight of the enzyme was determined by gel filtration on
a Superdex 200 HR 10/30 column (Pharmacia). Elution was performed with
KH2 PO4 buffer (50 mM, pH 7.0) containing 0.15 M NaCl at a flow rate of
 J. Gomes et al. / Enzyme and Microbial Technology 40 (2007) 969–975 971

0.5 ml min−1 . Fractions of 300 ␮l were collected and assayed for ␤-mannosidase Table 1
activity. Combination of different carbon and organic nitrogen sources in the growth
medium of T. aurantiacus and ␤-mannosidase activities obtained after 13 days
of cultivation at 47 ◦ C
2.7. Enzyme assays
Carbon source (15 g l−1 ) Nitrogen sourcea ␤-Mannosidase
The ␤-d-mannosidase activity was assayed according to Herr et al. [31] using (0.23 g N l−1 ) (nkat ml−1 )
200 ␮l of 3 mg ml−1 p-NP-␤-MP dissolved in sodium citrate buffer (50 mM, pH
Guar gum Soymeal 2.0
5), 200 ␮l of the enzyme solution and 400 ␮l sodium citrate buffer (50 mM,
Locust bean gum Soymeal 4.0
pH 5) at 50 ◦ C for 10 min. The reaction was stopped by the addition of 1.6 ml
Wheat straw (steam-treated)b Soymeal 1.1
Na2 CO3 (1 M) and the release of p-nitrophenol was monitored at 405 nm. Blank
solutions always contained all the components except the enzyme solution. ␣- Guar gum Pharmamedia 1.2
Mannosidase activity was assayed using p-NP-␣-MP as substrate. The assay Locust bean gum Pharmamedia 2.6
was performed as described for ␤-mannosidase. One nanokatal (nkat) of ␤- Wheat straw (steam-treated)b Pharmamedia 0.8
mannosidase activity was defined as that amount of enzyme required to catalyze
Guar gum Solulys 2.0
the release of 1 nmol p-nitrophenol s−1 under the assay conditions. ␤-Mannanase
Locust bean gum Solulys 3.4
activity was assayed at pH 5.0 and 50 ◦ C according to Bailey et al. [32] using
Wheat straw (steam-treated)b Solulys 1.0
a 0.5% solution of LBG as substrate. One nkat of ␤-mannanase activity was
defined as the amount of enzyme that produced 1 nmol of reducing sugars (as Mannosec Soymeal 0.9
mannose) in 1 s under the assay conditions. Reducing sugars were determined by Sugar beet pulp (starch free) Soymeal 3.0
the dinitrosalicylic acid method of Miller [33]. Soluble proteins were determined No carbohydrate Soymeal 0.25
by bicinchonic acid (BCA) protein assay method of Pierce (Rockford, Illinois, a Soymeal, Pharmamedia (cotton seed protein), and Solulys (spray-dried,
USA) using bovine serum albumin as standard. Absorbance at 280 nm was used
corn-steep liquor) contained 7.2%, 9.4% and 7.4% N, respectively.
to monitor the protein content in the column fractions. b Wheat straw was steam pre-treated as described by Gomes et al. [26].
c Mannose concentration was 20 g l−1 .
2.8. Determination of pH and temperature optima and stabilities

The pH optimum of the ␤-mannosidase was determined by measuring the

activity at pH values ranging from 2.0 to 6.5 using sodium citrate (pH 2.0–6.5,
100 mM) and glycine–HCl (pH 2.0–3.5, 100 mM) buffers. The effective pH
value of the reaction mixture was measured after mixing the enzyme solution
with the respective buffers. The temperature optimum of the ␤-mannosidase
was determined at different temperatures (50–80 ◦ C) according to the above
mentioned ␤-mannosidase assay procedure. For determination of the temper-
ature stability, the purified enzyme solution (in sodium citrate buffer, 50 mM,
pH 5.0) was incubated in screw capped tubes at 76 ◦ C for 2 h. At appropriate
time intervals sample tubes were withdrawn, cooled on ice and the residual
activity was measured. The pH stability was determined by diluting the puri-
fied enzyme with the buffer at different pH values (pH 2.0–6.5, sodium citrate
buffer, 100 mM; pH 7.0–8.0, phosphate buffer, 50 mM) and incubating the mix-
ture for 5 h at 50 ◦ C. The residual ␤-mannosidase activity was determined
subsequently.
2.9. Kinetic parameters
Kinetic parameters of T. aurantiacus ␤-mannosidase were determined using
reaction mixtures containing p-NP-␤-MP in sodium citrate buffer (100 mM, pH
3.0). The release of p-nitrophenol was measured using different initial substrate
concentrations (0.16–30 mM). All assays were performed in triplicate. Values
for the maximal reaction velocity (Vmax ) and Michaelis-Menten constant (Km )
were determined using the computer program, Sigma Plot (Version 7.0).
2.10. Transglycosylation and HPLC analysis
Transglycosylation activity of ␤-mannosidase was examined in a 0.7 ml final
reaction mixture. The enzyme (1 nkat ml−1 ) was incubated in 25 mM sodium
citrate buffer (pH 5.0) containing 31 mM p-NP-␤-MP as donor and 496 mM
methyl-␣-d-mannopyranoside as acceptor at 50 ◦ C. Negative controls were run
without adding ␤-mannosidase. Samples (100 ␮l) were taken in a screw capped
tube every 2–3 h, diluted with 100 ␮l of 10 mM KH2 PO4 buffer (pH 7.0) and the
enzymatic reaction was stopped by heating the mixture on a boiling water bath
for 1 min. The sugars in the cooled and filtered reaction mixture were analyzed by
HPLC. A Merck Hitachi LaChrom HPLC System equipped with refractive index
and UV detectors and an automatic sampler was used to separate products of
the reaction. The separation was performed using an Aminex HPX-87K column
(Bio-Rad) and KH2 PO4 -buffer (10 mM, pH 7.0) as an eluant at a flow rate of
0.6–0.8 ml min−1 at 85 ◦ C.
3. Results and discussion
3.1. Effect of carbon sources
To study the effect of carbon sources on the production
of ␤-mannosidase growth T. aurantiacus was grown in basal
medium [26] containing either no carbon source or one of the
five carbon sources (15 g l−1 ) and one of the three organic nitro-
gen (0.23 g N l−1 ) sources (Table 1). Among the five carbon
sources and three nitrogen (on equal nitrogen basis) sources
tested, the highest levels of mannanolytic activities, e.g., ␤-
mannanase (10 nkat ml−1 ), ␤-mannosidase (4 nkat ml−1 ) and
␣-mannosidase (0.4 nkat ml−1 ), were produced on LBG and
soymeal. Although structurally guar gum and locust bean gum
galactomannans are similar (except LBG has about two times
more mannose residues for every galactose residue [3]), guar
gum was a less effective inducer of ␤-mannosidase. Similar
observation was made for the mannanolytic enzyme production
by S. rolfsii on LBG and guar gum by Gübitz et al. [11]. Presence
of small amounts of mannan containing polysaccharides, e.g.,
glucomannans [34] in sugar beet pulp might have acted as a good
inducer for ␤-mannosidase. Further investigations using various
concentrations (5–20 g l−1 ) of LBG and soymeal (0.5–4.0 g l−1 )
indicated that maximum enzyme production was achieved in
the presence of 15 g l−1 LBG and 3.2 g l−1 soymeal (data not
shown). Henceforth the fungus was grown on LBG 15 g l−1 and
soymeal (3.2 g l−1 ). The fungus showed morphological differ-
ence during growth on monomer and polysaccharides, growing
in small pellet forms on mannose and in diffused form on galac-
tomannans. The ability of the fungus to grow well on soymeal in
the absence of any added carbon source indicates that soymeal
was most probably used as carbon and organic nitrogen sources.
Synthesis of low level of ␤-mannosidase in the absence of any
carbon source leads us to assume that ␤-mannosidase is a consti-
972 J. Gomes et al. / Enzyme and Microbial Technology 40 (2007) 969–975
Fig. 1. Time course of ␤-mannosidase production by Thermoascus aurantiacus,
grown on the medium supplemented with LBG or mannose or no added carbon
source. The data shown are averages of duplicate assays with S.D. within 10%
of the mean values.
tutive enzyme of T. aurantiacus. It had been speculated that low
level (1–3 nkat ml−1 ) production of ␤-mannosidase by T. auran-
tiacus on wheat straw [25] or Solka Floc [26] was due to the
absence of mannan containing polysaccharides in these carbon
sources. However, the maximum yield (4 nkat ml−1 ) obtained
by growing the fungus on LBG was only 1.33 times higher than
that obtained by growing the fungus on cellulosic substrate Solka
Floc [26]. Low level induction of ␤-mannosidase in this fungus
may be due to the fact that the primary function of this enzyme
may not be disaccharide hydrolysis. However, the induction of
higher levels of ␤-mannosidase by LBG, guar gum, copra meal,
konjak glucomannan and yeast mannan has been reported for
various microorganisms [11,13].
3.2. Time course of shake flask cultures
Progress of typical shake-flask cultures of T. aurantiacus on
LBG and mannose as carbon sources and without added carbon
source is shown in Fig. 1. In the presence of LBG the synthe-
sis of ␤-mannosidase commenced after about 18 h of cultivation,
increased slowly but steadily and reached a peak (3.6 nkat ml−1 )
at 210 h. In the absence of any added carbon source synthesis of
very little ␤-mannosidase, even after 210 h, was most probably
due to the utilization of the carbohydrates present in soymeal
and/or production of enzyme at constitutive level. Although the
fungus grew very well on mannose at an initial concentration of
20 g l−1 there was practically no ␤-mannosidase synthesis up to
90 h of cultivation. After the mannose concentration had dropped
to about 1 g l−1 at around 160 h ␤-mannosidase synthesis com-
menced, reaching a value of 0.95 nkat ml−1 at 210 h. Feldman et
al. [35] also observed that endoglucanase production by another
strain of T. aurantiacus was strongly repressed by glucose at
Table 2
Purification of ␤-mannosidase from the culture filtrates of Thermoascus aurantiacus, grown on LBG at 47 ◦ C
Purification steps Volume (ml) Protein (mg ml−1 )
Culture filtrate 320 0.62
Ethanol precipitate, soluble at pH 4.0 47 1.0
Cation-exchange chromatography 1 0.1
Fig. 2. SDS-PAGE of extracellular proteins soluble in sodium-formate buffer
(25 mM, pH 4.0) (lane 2) and ␤-mannosidase after purification by cation-
exchange chromatography on MonoS (lane 3). Molecular mass standards (lane 1,
LMW Std., Pharmacia Biosciences) are given in kDa. Staining with Coomassie
brilliant blue was used to visualize the protein bands.
initial high concentrations (10 g l−1 ) and the enzyme synthesis
began only when the glucose concentration was reduced to a
non-repressive level (ca. 2 g l−1 ).
3.3. Purification of the β-mannosidase
The isolation and purification of the ␤-mannosidase from
the culture filtrate of T. aurantiacus was achieved in three
steps. Precipitation of proteins with ethanol gave the highest
(78%) recovery of ␤-mannosidase activity compared to that
obtained with isopropanol (37%), acetone (27%) and ammo-
nium sulphate (33%) as precipitation agents. Proteins soluble
at pH 4.0 had high ␤-mannosidase activity and were free from
␤-mannanase activity. Residual proteins, soluble at pH 5.0, con-
tained only traces of ␤-mannosidase activity but high mannanase
activity. By means of ethanol precipitation procedure proteins
containing ␤-mannosidase were concentrated with concurrent
removal of other proteins. Thus, specific ␤-mannosidase activity
could be increased more than three-fold (16 nkat mg−1 protein).
By means of cation-exchange chromatography with MonoS
␤-mannosidase could further be purified to apparent homo-
geneity, raising the specific activity to 61 nkat mg−1 protein. A
single peak of ␤-mannosidase activity was detected in cation-
exchange chromatography and it appeared as a single band on
SDS-PAGE (Fig. 2). No ␣-mannosidase, ␤-glucosidase or ␣- or
␤-galactosidase activity could be detected in this fraction. An
11.4-fold purification of ␤-mannosidase together with a total
recovery of 29.9% was achieved by using only three purifica-
tion steps as summarized in Table 2.
Activity (nkat) Sp. activity (nkat mg−1 ) Purification (-fold) Yield (%)
960 5 100
752 16 3.2 78.3
6.1 61 11.4 29.9
 J. Gomes et al. / Enzyme and Microbial Technology 40 (2007) 969–975
3.4. Molecular mass and pI value
The purified ␤-mannosidase appeared homogenous as judged
by SDS-PAGE under denatured and non-denatured conditions
and gel filtration. This ␤-mannosidase was an acidic protein
with an isoelectric point of pI = 4.8, which is close to the pI val-
ues (4.7–5.0) reported for ␤-mannosidases from A. niger [6–8],
S. rolfsii [11] and T. reesei [12]. The molecular mass of the
␤-mannosidase as estimated by gel filtration corresponded to
99.9 kDa in line with the molecular mass of denatured enzyme
of 99 kDa as determined by SDS-PAGE. The average molecular
mass of the T. aurantiacus ␤-mannosidase was similar to those
(94–125 kDa) reported for the ␤-mannosidases from A. niger
[6,7], A. awamori [9], T. reesei [12], C. fimi [14] and T. fusca
(cloned in Streptomyces lividans) [15]. In contrast, the molecular
weights of ␤-mannosidases from S. rolfsii [11], P. furiosus [16]
and T. neapolitana [17] have been reported to be 58–65 kDa.
3.5. pH optimum and stability
The ␤-mannosidase was most active at low pH of 2.5–3.0
(Fig. 3a). This value corresponded well with the pH optima
(2.4–3.0) reported for the ␤-mannosidase from A. niger [8] and
S. rolfsii [11]. For most of the other ␤-mannosidases from fungi
like A. niger [7] and A. awamori [9,10] pH optima in the range
of 3.0–5.0 have been reported. Only few ␤-mannosidases, such
Fig. 3. Effect of pH on the activity (a) and stability (b) of the partially purified
␤-mannosidase from Thermoascus aurantiacus. The data shown are averages of
triplicate assays with S.D. within 10% of the mean values.
973
as those from C. fimi [14], T. fusca (expressed in S. lividans)
[15] and P. furiosus [16] have been reported to exhibit maxi-
mal activity at pH 7.0 or above. Although the T. aurantiacus
␤-mannosidase was most active at pH 2.5–3.0 it retained highest
activity (100%) after 5 h incubation at pH 5.9 and 50 ◦ C (Fig. 3b).
With the decrease or increase of pH value from 5.9 the stability of
enzyme decreased. Similar pH stability patterns were observed
for the cellulolytic and hemicellulolytic enzymes from this strain
of T. aurantiacus [25–27]. Good stability of the enzyme under
acidic conditions may be explained by the ability of the fungus
growing under acidic conditions. As the ␤-mannosidase shows
good stability under acidic conditions it may be used together
with other hemicellulolytic enzymes for application in chlorine
free softwood bleaching [11].
3.6. Temperature optimum and stability
As shown in Fig. 4a the temperature optimum of the ␤-
mannosidase was determined to be 76 ◦ C. This temperature
optimum of the ␤-mannosidase from the moderate thermophilic
fungus T. aurantiacus appeared distinctly higher than those
(60–70 ◦ C) reported for the ␤-mannosidases isolated from the
mesophilic fungi, like, A. niger [7,8], A. awamori K4 [10], T.
fusca [15]. This optimum temperature for the T. aurantiacus
␤-mannosidase activity is about 29 ◦ C higher than the opti-
mum growth temperature (47 ◦ C) of the fungus. On the other
Fig. 4. Effect of temperature on the activity (a) and stability (b) of ␤-
mannosidase from Thermoascus aurantiacus. The data shown are averages of
triplicate assays with S.D. within 10% of the mean values.
974 J. Gomes et al. / Enzyme and Microbial Technology 40 (2007) 969–975
hand, ␤-mannosidases from hyperthermophilic bacteria, such
as, P. furiosus [16] and T. neapolitana [17] are most active at
temperatures ranging from 87 to 105 ◦ C. The T. aurantiacus
␤-mannosidase lost half of its activity after 10 min incubation
at 76 ◦ C and pH 5.14 (Fig. 4b). This temperature stability of
the ␤-mannosidase was greater than that of the ␤-mannosidases
from A. niger [7,8], A. awamori K4 [10], T. reesei [13] and T.
fusca [15]. However, other enzymes, e.g., crude xylanase [25],
endoglucanase, ␤-glucosidase [26] and mannanase [27] from
the same fungal strains showed much higher temperature stabil-
ity than the purified ␤-mannosidase. The low heat sensitivity of
this ␤-mannosidase could be explained by its purity.
3.7. Kinetic properties
The Km and Vmax values of the T. aurantiacus ␤-
mannosidase for p-NP-␤-MP were determined to be 1.1 mM
and 61 nkat mg−1 . The turn over number (kcat ) was calcu-
lated to be 6.1 s−1 which resulted in a catalytic efficiency
(kcat /Km ) of 5.5 × 103 M−1 s−1 . Earlier investigators reported
Km values between 0.18 and 3.1 mM for ␤-mannosidases
obtained from different microorganisms, e.g., A. niger [6–8],
A. awamori [9], T. reesei [12], C. fimi [14], T. fusca [15],
P. furiosus [16] and T. neapolitana [17]. The kcat /Km for
the T. aurantiacus ␤-mannosidase (333 mM−1 min−1 ) was
lower than those values reported for ␤-mannosidases from A.
niger (848–913 mM−1 min−1 ) [7], T. neapolitana (1454 mM−1
min−1 ) [17] and P. furiosus (2400 mM−1 min−1 ) [16]. Since the
catalytic efficiency of the present ␤-mannosidase was signifi-
cantly low the primary role of the enzyme may not be disaccha-
ride hydrolysis.
3.8. Transglycosylation
Because of practical and theoretical importance the transg-
lycosylation activity of the purified ␤-mannosidase was inves-
tigated. Interestingly transglycosylation activity was observed
when the enzyme was incubated in the presence of p-
nitrophenyl-␤-d-mannopyranoside as donor and methyl-␣-d-
mannopyranoside as acceptor. The mannosyl group from p-NP-
␤-MP was transferred to methyl-␣-d-mannopyranoside. HPLC
chromatograms clearly showed the synthesis of a disaccharide
(with retention of configuration) that had HPLC retention time
similar to that of mannobiose. The amount of disaccharide was
found to increase up to 6 h, followed by a decrease, due to the
hydrolysis of the disaccharide. No transglycosylation reaction
occurred in the absence of the enzyme. Transglycosylation activ-
ity of ␤-mannosidase has also been reported for ␤-mannosidases
from A. niger [9], C. fimi [14] and T. fusca [15].
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