An α-L-fucosidase from Penicillium multicolor as a candidate enzyme for the synthesis of α (1→3)-linked fucosyl oligosaccharides by transglycosylation

Article abstract of DOI:10.1016/S0008-6215(98)00112-8

A new α-L-fucosidase was partially purified from the culture broth of Penicillium multicolor, which was available commercially as a freeze dried powder by the name of Lactase-P. This enzyme catalysed the transglycosylation of fucose residue of p-nitrophenyl-α-L-fucopyranoside to give α-L-Fuc-(1→3)-D-G1c or α-L-Fuc-(1→3)-D-GlcNAc regioselectiveiy. This enzyme was more stable in the organic co-solvents than the α-fucosidase from Aspergillus niger, which was also proposed previously by us as an enzyme to produce fucosyl oligosaccharides.

Full text of DOI:10.1016/S0008-6215(98)00112-8

Carbohydrate Research 309 (1998) 125±129
An ꢀ-l-fucosidase from Penicillium multicolor as
a candidate enzyme for the synthesis of ꢀ
(1!3)-linked fucosyl oligosaccharides by
transglycosylation
Katsumi Ajisaka *, Hiroshi Fujimoto, Mariko Miyasato
Meiji Institute of Health Science, Meiji Milk Products, Co., Ltd., 540 Naruda, Odawara 250, Japan
Received 5 January 1998; accepted 17 April 1998
Abstract
A new ꢀ-l-fucosidase was partially puri®ed from the culture broth of Penicillium multicolor,
which was available commercially as a freeze dried powder by the name of Lactase-P2. This
enzyme catalysed the transglycosylation of fucose residue of p-nitrophenyl-ꢀ-l-fucopyranoside to
give ꢀ-l-Fuc-(1!3)-d-Glc or ꢀ-l-Fuc-(1!3)-d-GlcNAc regioselectively. This enzyme was more
stable in the organic co-solvents than the ꢀ-fucosidase from Aspergillus niger, which was also
proposed previously by us as an enzyme to produce fucosyl oligosaccharides. # 1998 Elsevier
Science Ltd. All rights reserved
Keywords: ꢀ-l-fucosidase; Penicillium multicolor; ꢀ-l-Fuc-(1!3)-d-Glc; ꢀ-l-Fuc-(1!3)-d-GlcNAc; Trans-
glycosylation
1. Introduction
been developed [3±6]. However they were generally
cumbersome due to the multiple steps of protection
and deprotection processes.
A variety of biologically important glycoconju-
gates contain ꢀ-l-Fuc residue at the non-reducing
termini; for example lacto-N-fucopentaose I±III
in human milk [1] or H, Lex, and Leb determinants
in the blood-group substances [2]. The ꢀ-l-fuco-
syl residue is found with ꢀ-(1!2)-linkage to d-Gal
residue or with ꢀ-(1!3)-, ꢀ(1!4)-, or ꢀ-(1!6)-
linkage to d-GlcNAc residue in glycoconjugates.
To elucidate the biological functions of these fuco-
syl oligosaccharides, many chemical syntheses have
On the other hand, the enzymatic method, espe-
cially the use of glycosidases, is advantageous,
because of high stereo- and regioselectivity in the
transglycosylation [7,8]. In the previous report [9],
we have shown that the ꢀ-l-fucosidase [EC 3.2.1.5
1] from Aspergillus niger produced ꢀ-l-Fuc-(1!3)-
Glc or ꢀ-l-Fuc-(1!3)-GlcNAc in high yield and
high regioselectivity by transglycosylation using
pNP-ꢀ-Fuc as a donor. Several biologically inter-
esting fucosyl oligosaccharide derivatives have
been synthesized with the aid of this enzyme
[10,11].
* Corresponding author. Tel: 81-465-37-3661; fax: 81-
465-36-2776; e-mail: rxg0120@niftyserve.or.jp
0008-6215/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00112-8

126
K. Ajisaka et al./Carbohydrate Research 309 (1998) 125±129
However, the content of ꢀ-l-fucosidase activity
sponds to the amount of enzyme that produces
1 ꢁmol of para-nitrophenol per min. This solution
was used in the following experiments without fur-
ther puri®cation.
was not satisfactory for use in a practical produc-
tion of fucosyl oligosaccharides. Recently, we
found a new candidate ꢀ-l-fucosidase of microbial
origin exhibiting similar potentials in regioselec-
tivity and yield in the transglycosylation. The
enzyme was obtained from the culture broth of
Penicillium multicolor, whose powdered prepara-
tion was commercially available. In the present
report, we describe the characterization of this
enzyme and the synthesis of ꢀ1±3 linked fucosyl
disaccharides by transglycosylation.
Measurement of molecular weight.ÐThe enzyme
solution (10 ꢁL) was applied to HPLC with TSK
G-3000 SW column and eluted with 0.1 M sodium
phosphate bu€er (pH 7.4) containing 0.1 M sodium
sulfate. When the column was eluted at a ¯ow rate
of 0.5 mL/min, the ꢀ-l-fucosidase activity was
found at 15.5 min. From the calibration curve
obtained by using the protein kit of Sigma, the
molecular weight was estimated as ca. 180 kD.
E€ect of pH and temperature.ÐThe optimum
pH was measured using 0.1 M acetate bu€er and
0.1 M phosphate bu€er. Enzyme solution (0.1 mL)
was diluted with 1.4 mL of the bu€er solution at
various pHs, and the activity was measured by
using pNP-ꢀ-Fuc. For the measurement of pH
stability, 0.1 M glycine bu€er, acetate bu€er, and
phosphate bu€er were used. The enzyme solutions
at various pHs were kept at 25 ꢀ for 24 h. Then
0.1 mL of the enzyme solution was mixed with
1.4 mL of 0.1 M phosphate bu€er (pH 5.3) and the
ꢀ-l-fucosidase activity was measured.
2. Experimental
Materials.ÐLactase-P2, a powdered culture
broth of P. multicolor, was purchased from K. I.
Chemical Industry Co., Ltd., (Shizuoka, Japan).
pNP-ꢀ-Fuc was a product of Sigma. ꢀ-l-Fuc-
(1!2)-Gal and ꢀ-l-Fuc-(1!6)-GlcNAc were pur-
chased from Funakoshi Co., Ltd. (Tokyo).
Analytical Methods.ÐHPLC was carried out
using a AKTA-design system (Pharmacia) with
TSK G-3000 SW, Waters AccQ Tag, or Asahipak
NH2P50 column. Otherwise a Dionex Bio-LC sys-
tem was used with CarboPac PA-1 column and
50 mM sodium hydroxide solution as eluent. The
13C NMR spectrum was recorded on a Varian
Inova 500 spectrometer. Chemical shifts are
expressed in ppm relative to internal acetonitrile
(1.27 ppm) in D2O.
Partial puri®cation of a-l-fucosidase from P.
multicolor.ÐPowder (1 g) of Lactase-P2 (ꢀ-l-
fucosidase activity; 3.9 units/g) was dissolved in
10 mL of 1 mM sodium phosphate bu€er (pH 6.8)
and dialyzed against the same bu€er. The enzyme
solution was applied to Bio-Gel HTP-gel column
(BioRad Labs, 2.6Â11 cm), eluted with a gradient
of 1 mM to 400 mM sodium phosphate bu€er (pH
6.8) with a ¯ow rate of 0.5 mL/min. Fractions
(1 mL each) were collected and 10 ꢁL of each frac-
tion in 90 ꢁL of 0.1 M sodium acetate bu€er (pH
5.0) was incubated with 5 mM Fuc-ꢀ-pNP (50 ꢁL)
for 45 min at 37 ꢀC. The absorbance at 410 nm was
measured and plotted as a relative intensity of ꢀ-l-
fucosidase activity. Fractions containing ꢀ-l-fuco-
sidase activity (Fr. 28±34, 3.5 mL) were collected
and concentrated to ca. 0.5 mL using Ultrafree
CL2 (Millipore, molecular cut-o€ 30 kD). The
activity of the enzyme solution was 4.4 units/mL
(41.3 units/g). Here, 1 unit of ꢀ-l-fucosidase corre-
The thermal stability was also measured by
heating 0.44 unit of enzyme in 0.1 M sodium acet-
ate bu€er (pH 5.0, 1 mL) at various temperature
for 30 min. Then the remaining activity was mea-
sured at 37 ꢀC.
E€ect of organic solvents.ÐThe ®nal concentra-
tion of the organic solvent was made to 10, 20, and
30% in 0.1 M sodium acetate bu€er (pH 5.0) con-
taining 0.1 unit of enzyme. Each solution was kept
ꢀ
for 3 h at 37 C, then the remaining activity was
measured.
Substrate speci®city.ÐEach disaccharide solu-
tion (0.1 mg/mL in 0.1 M acetate bu€er (pH 5.0),
900 ꢁL) was mixed with 100 ꢁL of enzyme solution
ꢀ
(0.01 unit), and incubated at 37 C. An aliquot
(50 ꢁL) was withdrawn at an appropriate time
intervals and ®ltered through a membrane of
Ultrafree MC2 (Millipore, molecular cut-o€
30 kD). The peak area of the remaining dis-
accharide in HPLC (CarboPac PA-1) was obtained
by integration and plotted against time.
Synthesis of a-l-Fuc-(1!3)-d-GlcNAc by trans-
glycosylation using partially puri®ed enzyme.Ð
d-GlcNAc (500 mg) and pNP-ꢀ-Fuc (100 mg) were
dissolved in 8.5 mL of 0.1 M sodium acetate bu€er
(pH 5.0) containing 2 mL DMSO, then 1.5 mL of
partially puri®ed enzyme (6.2 units) was added. The

K. Ajisaka et al./Carbohydrate Research 309 (1998) 125±129
127
ꢀ
ꢀ
solution was incubated at 37 C, and the reaction
was monitored by HPLC with AccQ Tag column.
When the peak of pNP-ꢀ-Fuc disappeared in HPLC
the reaction was stopped by heating the solution for
5 min in a boiling water. In the case of the present
experiment, the reaction was stopped at 4 h. The
reaction mixture was applied onto the activated
carbon column (2.7Â50 cm). The column was
washed with 500 mL of water and subsequently
eluted with a gradient of water (1 L)±30% methanol
(1 L). The sugar was detected by phenol±sulfuric
acid method [12], and the fractions containing dis-
accharide were collected and concentrated by
vacuum evaporator to give 63.5 mg of syrup. The
product a€orded 13C NMR chemical shifts (100.8,
101.0, 8 1.6, 79.3, 62.0, 61.9, 16.7, 16.3 ppm) which
supported the disaccharide to be ꢀ-l-Fuc-(1!3)-
GlcNAc [9]. The yield was 49.3%.
Synthesis of a-l-Fuc-(1!3)-d-Glc by trans-
glycosylation using crude enzyme.Ðd-Glc (500 mg)
and pNP-ꢀ-Fuc (100 mg) were dissolved in 10 mL
of 0.1 M sodium acetate bu€er (pH 5.0) containing
10% DMF. Lactase-P2 (100 mg) was added to ꢀthe
solution and the mixture was incubated at 37 C.
for 20 h. By the puri®cation with activated carbon
column chromatography similarly to ꢀ-l-Fuc-
(1!3)-GlcNAc puri®cation., 35.2 mg of ꢀ-l-Fuc-
(1!3)Glc was isolated (27.9% yield). The structure
was con®rmed by comparing the chemical shifts of
the characteristic peaks in 13C NMR spectrum
(100.4, 96.6, 93.0, 83.8, 80.8, 61.7, 61.5 ppm) with
those of the literature data [9].
accharides was generally performed at 37 C, this
enzyme was stable enough for the reaction.
As aqueous bu€er is not a good solvent for Fuc-
ꢀ-pNP, therefore the addition of organic co-solvent
Fig. 1. Partial puri®cation of ꢀ-l-fucosidase from crude P.
multicolor broth by Bio-Gel HTP column chromatography.
(^); absorbances at 280 nm, (*); absorbance at 410 nm after
the treatment of 10 ꢁL of each fraction in 90 ꢁL of 0.1 M
sodium acetate bu€er (pH 5.0) with 5 mM Fuc-ꢀ-pNP (50 ꢁL)
ꢀ
for 45 min at 37 C.
3. Results and discussion
For the puri®cation of ꢀ-l-fucosidase from P.
multicolor, Bio-Gel HTP column chromatography
was performed and the result is shown in Fig. 1.
Fortunately, ꢀ-l-fucosidase activity appeared at
the position where the other proteins were not
eluted. Therefore the activity was enhanced to
10-fold by one step puri®cation with ca. 56%
recovery.
In the examination of the e€ect of pH, the max-
imum activity was observed at pH 5 as shown in
Fig. 2(a). Fig. 2(b) shows that the enzyme was
stable in the pH range of 3ꢁ7. In the experiment of
theꢀthermal stability, the enzyme was stable up to
50 C (data not shown). The thermal stability was
almost the same as that of A. niger ꢀ-l-fucosidase.
As the transglycosylation to synthesize dis-
Fig. 2. Curves for optimum pH (A) and pH stability (B). (~),
(*) and (&) represent the activities measured in 0.1 M glycine
bu€er, acetate bu€er, and phosphate bu€er, respectively.

128
K. Ajisaka et al./Carbohydrate Research 309 (1998) 125±129
Table 1
The residual ꢀ-l-fucosidase activity in various organic
cosolvents
peak corresponding to the regio-isomer around the
peak of ꢀ-l-Fuc-(1!3)-d-GlcNAc (peak C) in
Fig. 3, suggesting that there is no other isomer than
ꢀ-l-Fuc-(1!3)-d-GlcNAc. HPLC using other col-
umn such as CarboPac PA-1 also showed only a
peak of ꢀ-l-Fuc-(1!3)-d-GlcNAc in the dis-
accharide region (data not shown). Actually after
the fractions corresponding to the disaccharide
elution in the activated carbon column chromato-
graphy were corrected and concentrated to dry-
ness, NMR was measured to give a spectrum of
pure ꢀ-l-Fuc-(1!3)-d-GlcNAc.
Organic solvent
Concentration
(v/v%)
Residual activity %
A. niger P. multicolor
10%
20%
30%
10%
20%
30%
10%
20%
30%
97
68
20
100
0
100
100
58
100
71
1
100
55
DMSO
DMF
0
96
14
0
Dioxane
0
The substrate speci®city in the hydrolysis reac-
tion was examined qualitatively at ®rst by using
ꢀ-l-Fuc-(1!2)-d-Gal, ꢀ-l-Fuc-(1!3)-d-GlcNAc,
and ꢀ-l-Fuc-(1!6)-d-GlcNAc. This enzyme
hydrolysed all the above disaccharides (data not
shown). This result was incompatible with the
result that only the ꢀ-l-Fuc-(1!3)-d-GlcNAc was
synthesized by transglycosylation using GlcNAc as
acceptor (Fig. 3). In this reaction, ꢀ-l-Fuc-(1!6)-
d-GlcNAc should be synthesized as it was a good
substrate for this enzyme, since according to our
previous results [8,9] the disaccharides hydrolyzable
by one glycosidase should be formed by the trans-
glycosylation. In addition, when d-Gal was used as
an acceptor, no transglycosylated product was
obtained. Then we compared relative hydrolysis rate
of pNP-ꢀ-Fuc and these disaccharides. As shown in
Fig. 4, ꢀ-(1!3)-linked disaccharide was hydrolysed
rather slower than ꢀ-(1!6)-isomer and ꢀ-(1!2)-
isomer which were hydrolysed with a similar rate
to pNP-ꢀ-Fuc. Therefore it can be explained that
is necessary to raise the molar ratio of donor. In
that case, the stability of enzyme toward organic
solvents becomes signi®cant. Therefore, the stabi-
lity of the present enzyme and ꢀ-l-fucosidase from
A. niger was examined towards DMSO, DMF, and
dioxane. Table 1 shows the relative residual activ-
ities after 3 h. The present enzyme was stable in
20% DMSO solution, while ꢀ-l-fucosidase from
A. niger lost 33% of its activity at the same condi-
tion. For all the experiments on three organic sol-
vents, the stability of ꢀ-l-fucosidase from P.
multicolor was superior to that from A. niger.
Representative HPLC chart of the transglycosy-
lation for the synthesis of ꢀ-l-Fuc-(1!3)-d-
GlcNAc is demonstrated in Fig. 3. There is no
Fig. 3. HPLC of the reaction mixture after 3 h from the start
of the reaction. Column:Asahipak NH2-P50 (4.6ꢂÂ250 mm);
Elution: 80% acetonitrile; ¯ow 0.8 mL/min; Detection:
210 nm. Peaks A, B, C, D, and E are assigned as solvents and
pNP-ꢀ-Fuc, Fuc, ꢀ-l-Fuc-(1!3)-d-GlcNAc, GlcNAc, and
p-nitrophenol, respectively.
Fig. 4. Relative rate of hydrolysis of pNP-ꢀ-Fuc (~), ꢀ-l-
Fuc-(1!2)-d-Gal (^), ꢀ-l-Fuc-(1!3)-d-GlcNAc (*), and ꢀ-
l-Fuc-(1!6)-d-GlcNAc (&) by the present enzyme.

K. Ajisaka et al./Carbohydrate Research 309 (1998) 125±129
Acknowledgements
129
ꢀ-l-Fuc-(1!6)-d-GlcNAc or ꢀ-l-Fuc-(1!2)-d-
Gal once formed by transglycosylation would be
hydrolysed as soon as they were formed, because
the formation rate of ꢀ-l-Fuc-(1!6)-d-GlcNAc
or ꢀ-l-Fuc-(1!2), which was equal to the hydro-
lysis rate of pNP-ꢀ-Fuc, was almost the same as
the hydrolysis rate of these disaccharides. In
contrast, ꢀ-l-Fuc-(1!3)-d-GlcNAc, whose hydro-
lysis rate was slow, accumulated in the reaction
mixture.
This work was performed as a part of the
Research and Development Projects of Industrial
Science and Technology Program supported by
NEDO (New Energy and Industrial Technology
Development Organization).
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In the reaction using crude enzyme, the product
was also unique and the reaction was also con-
®rmed regioselective.
Although we have previously proposed the ꢀ-l-
fucosidase from A. niger to be suitable for the
production of ꢀ-(1!3)-linked fucosyl disac-
charides regioselectively, the ꢀ-l-fucosidase from
P. multicolor also exhibited a similar potential in
transglycosylation reaction. The yield of ꢀ-l-Fuc-
(1!3)-d-GlcNAc using the present enzyme (49%)
was almost equal to A. niger ꢀ-l-fucosidase (58%).
However, P. multicolor produced about ®ve times
more ꢀ-l-fucosidase than A. niger. Moreover, P.
multicolor ꢀ-l-fucosidase was more stable in
organic solvent than the A. niger ꢀ-l-fucosidase.
The enhanced stability in organic solvent is not
only e€ective in dissolving pNP-ꢀ-Fuc to a higher
concentration, but also e€ective in decreasing the
amount of enzyme, since the enzyme is tolerable in
the extended reaction time.
[11] A. Vetere, C. Galateo, and S. Paoletti, Biochem.
Biophys. Res. Commun., 234 (1997) 358±361.
[12] H. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers,
and F. Smith, Anal. Chem., 28 (1956) 350±356.
In conclusion, P. multicolor was superior to A.
niger in the production of ꢀ-l-fucosidase for
synthesis ꢀ-(1!3)-linked fucosyl oligosaccharides.