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Applied and Environmental Microbiology, January 2000, p. 252-256, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Purification and Characterization of
2,6-
-D-Fructan 6-Levanbiohydrolase from
Streptomyces exfoliatus F3-2
Katsuichi
Saito,1
Kazuya
Kondo,1
Ichiro
Kojima,2
Atsushi
Yokota,3,* and
Fusao
Tomita1
Laboratory of Applied
Microbiology1 and Laboratory of
Microbial Resources and Ecology,3 Research Group
of Molecular Bioscience, Division of Applied Bioscience, Graduate
School of Agriculture, Hokkaido University, Sapporo 060-8589, and
Central Technical Research Laboratory, Nippon Mitsubishi
Oil Corporation, Yokohama 231-0815,2 Japan
Received 8 July 1999/Accepted 27 October 1999
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ABSTRACT |
Streptomyces exfoliatus F3-2 produced an extracellular
enzyme that converted levan, a 
-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a
single band on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). The molecular weights of this enzyme were
54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the
monomeric structure of the enzyme. The isoelectric point of
the enzyme was determined to be 4.7. The optimal pH and temperature of
the enzyme for levan degradation were pH 5.5 and 60°C, respectively.
The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in
an exo-acting manner. It was also found that this enzyme catalyzed
hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and
1-fructosylnystose by liberating fructose. Thus, this enzyme appeared
to hydrolyze not only -2,6-linkage of levan, but also
-2,1-linkage of fructooligosaccharides. From these data, the enzyme
from S. exfoliatus F3-2 was identified as a novel
2,6--D-fructan 6-levanbiohydrolase (EC 3.2.1.64).
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INTRODUCTION |
It has been reported that
oligosaccharides appear to exhibit various physiological functions
according to their structures. As the functions of the oligosaccharides
are recognized, the production methods become important. An attempt to
synthesize a large number of novel oligosaccharides has been made by
using sugar hydrolases or sugar transferases. We have been studying the
effective production of novel oligosaccharides (11, 13-15,
19-22) from such unused natural fructans as levan and inulin by
the microbial polysaccharide-degrading enzymes. Also we confirmed that
two of these oligosaccharides, di-D-fructose-1,2':2,3'-dianhydride (DFA III)
(17) and di-D-fructose-2,6':6,2'-dianhydride (DFA IV) (12), had effects on increasing calcium absorption in the small intestine of rats, which could be expected to have practical applications in such fields as treatment with functional food
or medicine to prevent osteoporosis.
In the course of these studies, Streptomyces exfoliatus F3-2
was isolated as a strain producing a levan-degrading enzyme (LDE) that
effectively produces levanbiose from levan (21). Levan is a
polymer consisting of -2,6-linked fructose residues and found in
monocotyledons as a reserve carbohydrate or synthesized by microbial
levansucrase (EC 2.4.1.10) from sucrose (3, 4). In contrast
to a plant levan, a microbial levan generally has a lot of branching
points at the C-1 position, except for a levan from Serratia
levanicum (4, 5). In our previous study
(21), we demonstrated that the combination of the LDE from
S. exfoliatus F3-2 and levan from S. levanicum
was quite efficient for the large-scale preparation of levanbiose, with a maximum production yield of levanbiose of 84% (wt/wt) from 50 mg of
levan per ml. The linear nature of the levan molecule from S. levanicum seems to contribute to this high yield.
To date, three types of microbial LDEs have been reported: levanase (EC
3.2.1.65), which hydrolyzes levan into fructose (1);
2,6--D-fructan 6-levanbiohydrolase (EC 3.2.1.64), which hydrolyzes levan into levanbiose (2); and levan
fructotransferase, which produces DFA IV from levan (11, 13,
18). The enzyme from Pseudomonas sp. was the only
enzyme reported as a 2,6--D-fructan 6-levanbiohydrolase.
However, the enzyme was not purified completely, and the enzymological
properties of levanbiose-producing LDE are still unclear
(2).
Thus the purification and characterization of the levanbiose-producing
LDE from S. exfoliatus F3-2 were conducted in order to
investigate the properties of the enzyme and to obtain more information
on the effective production of levanbiose.
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MATERIALS AND METHODS |
Culture conditions for S. exfoliatus F3-2 and
preparation of crude LDE.
The culture conditions and media for
S. exfoliatus F3-2 were those of our previous report
(21), except that the optimized LDE production medium was
used for LDE production. The optimized LDE production medium consisted
of (per liter) 10 g of levan (prepared from culture of S. levanicum), 2 g of NH4NO3, 0.5 g
of MgSO4 · 7H2O, 0.5 g of KCl,
0.5 g of KH2PO4, 0.01 g of
FeSO4 · 7H2O, 0.5 g of meat extract
(Wako Pure Chemical Industries Ltd., Osaka, Japan), 20 g of
CaCO3, and NaOH to adjust the pH to 7.0. Crude LDE was
prepared as reported previously (21) by culturing strain F3-2 in optimized LDE production medium for 36 h at 32°C.
Chemicals.
Levan was prepared from a culture of S. levanicum (5) by the method described previously
(21). Levanbiose was enzymatically prepared from levan by
using the LDE from S. exfoliatus F3-2 (21). For
preparation of levantriose, 2 g of levan was partially hydrolyzed with 10 ml of 0.01 N HCl at 80°C for 30 min. Hydrolysates were then
put on an activated charcoal column (2.6 by 100 cm) washed with
deionized water, and sugars in the column were eluted with deionized
water. Levantriose-containing fractions (20 ml each) were detected by
thin-layer chromatography (TLC), combined, concentrated under reduced
pressure, and lyophilized. TLC was done by using a silica gel plate
(silica gel 60; Merck) with a solvent system of
1-butanol-2-propanol-water-acetic acid (7:5:4:2 [vol/vol]) and
developed at 12 cm twice. For detection of spots, the plate was sprayed
with a reagent consisting of
p-anisaldehyde-H2SO4-ethanol (1:1:18 [vol/vol]), dried, and kept at 120°C until spots appeared (19). Fructooligosaccharides (i.e., 1-kestose, nystose, and 1-fructosylnystose) were gifts from Meiji Seika Kaisya, Ltd., Tokyo,
Japan. Protein molecular weight markers were obtained from Roche
Diagnostics GmbH, Mannheim, Germany.
Enzyme assay.
The enzyme activity was assayed under the
standard LDE assay conditions as follows. The reaction mixture
contained 50 mM sodium phosphate buffer (pH 5.5) and 10 mg of levan per
ml, and the enzyme solution was diluted appropriately in a total volume
of 1 ml. The mixture was incubated at 40°C for 10 min and then heated
in boiling water for 5 min to stop the enzyme reaction. Next to check the presence of the LDE activity, sugars in the reaction mixture were
analyzed qualitatively by TLC. After that, for quantitative determination of enzyme activity, the reducing sugars in the reaction mixtures were assayed by the Somogyi-Nelson method (16). One unit of enzyme activity was defined as the amount of the enzyme that
produced 1 µmol of reducing sugars as fructose per min under these
assay conditions. The protein concentration was determined by the
methods of Lowry et al. (7). Unless otherwise mentioned, this method was used for the LDE assay in the experiments.
Purification of LDE.
All purification processes were done at
4°C. Crude LDE (1,000 ml [1,789 U]) was concentrated in dialysis
tubes with polyethylene glycol 20,000 for several hours. To the
concentrated solution, solid ammonium sulfate was added to give 60%
saturation. The precipitate formed was collected, dissolved in 20 ml of
0.1 M sodium phosphate buffer (pH 6.0), and dialyzed against
equilibration buffer for column chromatography. Anion-exchange column
chromatography was done on a DEAE-Toyopearl 650M column (2.2 by 18 cm)
(Tosoh Co., Ltd., Tokyo, Japan) equilibrated with 10 mM sodium
phosphate buffer (pH 6.0). The enzyme was eluted with a linear gradient
of 0 to 0.15 M NaCl in the same buffer, and fractions were collected at 7.5 ml each. The LDE activity-positive fractions which gave single band
by SDS-PAGE were combined and were used as the purified sample. SDS-PAGE was done on a 10% polyacrylamide gel by the method of Laemmli
(6). The gel was stained with silver stain.
Estimation of molecular weight of LDE.
A TSKgel
G3000SWXL high-performance liquid chromatography (HPLC)
column (7.8 by 300 mm) (Tosoh Co., Ltd.) was used with 10 mM sodium
phosphate buffer (pH 6.0) containing 0.2 M NaCl for equilibration and
elution buffer at a flow rate of 0.8 ml/min. The eluted proteins were
fractionated by monitoring A280, and the LDE
activity-positive fractions were located.
Isoelectric point of LDE.
Isoelectric focusing was done by
using a Rotofor apparatus (Bio-Rad Laboratories, Hercules, Calif.) with
Bio-Lyte 3/5 (Bio-Rad Laboratories), and the LDE activity-positive
fractions were located.
Reaction products from levan by the LDE.
The degradation
reaction was performed under the same conditions as those of the
standard LDE assay conditions, except that the final concentration of
the purified LDE was 0.8 U/ml and an incubation time of up to 24 h
was employed. At various time points, the reaction mixtures were heated
in boiling water for 5 min to stop the enzyme reaction, and the sugars
were analyzed by TLC and HPLC. HPLC analyses were done under the
conditions described previously (YMC-Pack ODS-AQ column, 6 by 250 mm
[YMC Co. Ltd., Kyoto, Japan]; mobile phase, water; refractive index
detector) (20) to determine the concentration of the
reaction products by using fructose and levanbiose as standards.
Mode of degradation by LDE.
To address whether LDE
hydrolyzes levan in an endo- or exo-acting manner, the degradation
reaction was conducted under standard reaction conditions, except that
purified LDE concentrations of 0.04 and 0.4 U/ml and a reaction time of
up to 24 h were employed. Sugars in the reaction mixtures were
analyzed at appropriate intervals quantitatively by the Somogyi-Nelson
method and HPLC, both by using levanbiose as the standard and
qualitatively by TLC. The ratio of the amount of reducing sugars versus
the amount of levanbiose determined by HPLC was then calculated. This
value would be 1 if the reducing sugar in the reaction mixture
consisted solely of levanbiose, while a value of higher than 1 would be expected if the reducing sugars other than levanbiose were
produced in the reaction mixture. The calculated values were correlated
with the distribution of sugars detected by TLC analyses of the
reaction mixtures. The mode of action was deduced from the values of
the initial reaction products, assuming that a value around 1 results from an exo-acting manner and a value higher than 1 results from an
endo-acting manner.
Substrate specificity of LDE.
For substrate specificity of
the purified LDE, 10 mg of levan per ml as a substrate under the
standard LDE assay conditions was replaced with the same amount of
various kinds of sugars, and a final enzyme concentration of 0.8 U/ml
was employed. The substrates and the reaction products after incubation
for up to 4 h were analyzed by TLC and HPLC as described above.
The percentages of degradation of the substrates were determined by
comparing the amounts of the decreased substrates measured by HPLC
against the initial amounts of the substrates. In the substrate
specificity experiments, monosaccharides produced in the reaction
mixture were analyzed by using the F-kit (food analysis
D-glucose/D-fructose; Roche Diagnostics GmbH).
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RESULTS AND DISCUSSION |
Purification of LDE.
According to the methods described in
Materials and Methods, the LDE was purified about 50-fold with a
recovery of 31% from the culture supernatant, and the specific
activity of the purified LDE was 105.0 U/mg. LDE gave a single band on
SDS-PAGE, as shown in Fig. 1A. The
molecular weights of the purified LDE were estimated to be 54,000 by
SDS-PAGE and 60,000 by gel filtration with Sephacryl S-200 SF (Fig.
1B). Thus, the enzyme from S. exfoliatus F3-2 was considered
to be a monomer. Since the two already reported levanbiose-producing LDEs from Streptomyces sp. strain 7-3 (10) and
Arthrobacter sp. strain 51A (8) are monomeric
enzymes, the LDE from S. exfoliatus F3-2 was found to be
structurally similar to these LDEs.
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FIG. 1.
SDS-PAGE and gel chromatography of the purified LDE. (A)
Lanes 1, marker proteins; 2, purified LDE. The marker proteins were
fructose-6-phosphate kinase (Mr, 85,200),
glutamate dehydrogenase (Mr, 55,600), aldolase
(Mr, 39,200), and triosephosphate isomerase
(Mr, 26,600). (B) Plots of the logarithmic
molecular weight versus retention time in gel filtration. The position
of the sample enzyme is shown by the solid circle. a, bovine serum
albumin (Mr, 68,000); b, egg albumin
(Mr, 45,000); c, chymotrypsinogen A
(Mr, 25,000); d, cytochrome c
(Mr, 12,500).
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pH and thermal properties.
The isoelectric point of the
purified LDE was estimated to be 4.7. The effects of pH and temperature
on both the reaction and stability of the purified LDE were
examined, and the results are shown in Fig.
2. The maximal enzyme activities were
obtained at pH 5.5 (Fig. 2A) and at 60°C (Fig. 2B). More than 90% of
the enzyme activities were found to remain in the pH range 3.5 to 8.0 (Fig. 2A) or at temperatures of up to 50°C (Fig. 2B).
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FIG. 2.
pH and thermal properties of the purified LDE. For
determination of optimal reaction pH (A [ ,  ,  ]), the
standard LDE assay was employed, except that the phosphate buffer was
replaced when necessary. The buffers used were 50 mM citric acid-sodium
citric acid buffer (), sodium phosphate buffer (), or boric
acid-sodium tetraborate buffer (). For pH stability (A [ ,  ,
 ]), the purified enzyme was mixed with the same volume of 0.1 M
different kinds of buffers as used for the determination of optimal pH.
After the incubation at 4°C for 24 h, the enzyme was diluted
with 4 volumes of 0.1 M sodium phosphate buffer (pH 5.5) and used as
the treated enzyme. The optimal reaction temperature (B [ ]) was
determined under standard LDE assay conditions, but at various
temperatures. Temperature stability (B [ ]) was tested by heating
the purified enzyme at various temperatures for 20 min. For
determination of optimal reaction pH and temperature, the maximum
activities obtained under the conditions tested were taken as 100%.
For estimation of pH and temperature stabilities, the activities of the
untreated enzyme were taken as 100%.
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Reaction products from levan.
The reaction products from levan
with purified LDE were examined as described in Materials and Methods.
As shown in Fig. 3A, levanbiose was
detected as a major product from the beginning of the reaction.
Levanoligosaccharides having a degree of polymerization more than 3 are
transiently detected, with a decrease in levan, especially after
reaction for 1 to 2 h. The concentration of levanbiose reached its
maximum, 9.3 mg/ml, after reaction for 8 h, and then degraded
slowly into fructose, as can be seen in Fig. 3B.
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FIG. 3.
TLC and HPLC analyses of the reaction products from
levan with purified LDE. The enzyme reaction and the analyses of the
sugars in the reaction mixtures by TLC and HPLC were done as described
in Materials and Methods. (A) TLC analysis. S, partial HCl hydrolysates
of levan. F, fructose; F2, levanbiose; F3,
levantriose; F4, levantetraose; L, levan. (B) HPLC analysis
of the reaction mixtures shown in panel A. , levanbiose; ,
fructose.
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Mode of action.
The mode of action was investigated as
described in Materials and Methods. Figure
4A shows the results obtained with the
reaction mixture containing the purified LDE at 0.04 U/ml, where the
HPLC measurement revealed that the degradation reaction yielded 2.3 mg
of levanbiose per ml from 10 mg of initial levan per ml after reaction
for 24 h. Thus, this reaction was designed to represent the
initial event of the degradation reaction. The results indicated that
the ratio (amount of reducing sugar/amount of levanbiose) at each
reaction time until 24 h appeared to be almost constant and was
close to the theoretical value of 1. TLC analysis of these reaction
mixtures showed that only levanbiose was detected as the reaction
product. The same results were also confirmed by HPLC analysis (data
not shown). From these results, the enzyme was judged to degrade levan
into levanbiose in an exo-acting manner.
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FIG. 4.
Ratio of the amount of reducing sugar to the amount of
levanbiose in the reaction mixture for levan degradation with purified
LDE. The purified LDE concentrations in the reaction mixtures were 0.04 U/ml for panel A and 0.4 U/ml for panel B. The TLC analysis of the
reaction mixture at each time point is shown right under the graph.
Abbreviations for TLC are defined in the legend to Fig. 3. *, ratio
of the amount of reducing sugar as levanbiose determined by the
Somogyi-Nelson method versus the amount of levanbiose determined by
HPLC.
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On the other hand, when the purified LDE was increased up to 0.4 U/ml,
the reaction proceeded rapidly, and the concentration
of levanbiose
reached 8.7 mg/ml after 24 h of incubation. Thus,
these reaction
conditions were aimed at investigating the event
occurring when the
reaction had proceeded almost completely. As
shown in Fig.
4B, the
ratios increased from 1 over time, and especially
at time points later
than 4 h, the ratios reached up to 1.4 until
24 h of
incubation. In agreement with these findings, TLC analysis
indicated
the presence of fructose and/or glucose and oligosaccharides
other than
levanbiose, suggesting that the ratio can be used as
a measure for the
formation of a reducing sugar other than
levanbiose.
2,6-
-
D-Fructan 6-levanbiohydrolase (EC 3.2.1.64) has
been defined as the enzyme that specifically cleaves levan into
levanbiose
(
2). Therefore, the levanbiose-producing LDE from
S. exfoliatus F3-2 was clearly judged to be
2,6-
-
D-fructan 6-levanbiohydrolase.
Substrate specificity.
The results of the enzyme reaction to
various kinds of sugars other than levan as a substrate are summarized
in Table 1. All reactions were carried
out for 4 h under the conditions described in Materials and
Methods. As has been suggested from the results shown in Fig. 3,
levanbiose was confirmed to be the smallest substrate for this enzyme,
although the hydrolyzing activity was low. This slow rate of levanbiose
degradation seems to result in efficient production of levanbiose from
levan, as established in our previous study (21).
Levantriose was degraded efficiently into levanbiose and fructose.
Thus, the smallest substrate for levanbiose formation by the enzyme
appeared to be levantriose.
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TABLE 1.
Reaction of purified 2,6--D-fructan
6-levanbiohydrolase from S. exfoliatus F3-2 to various kinds
of sugars
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A particularly interesting feature of this enzyme is the fact that it
can hydrolyze
-2,1-fructosyl linkages of fructooligosaccharides,
such as 1-kestose, nystose, and 1-fructosylnystose, with substantial
reaction rates comparable to that for levantriose, as described
in
Table
1. Figure
5 shows the results of
TLC analyses of these
reactions. From Table
1 and Fig.
5, it appears
that the percentage
of degradation of each substrate increased as the
degree of polymerization
of the substrate increased. However,
practically no degradation
reaction was detected when inulin was used
as the substrate under
the same reaction conditions (data not shown).
Monosaccharides
produced from these fructooligosaccharides were
identified as
consisting mainly of fructose by enzymatic determination
with
the F-kit (data not shown). From these results, it was confirmed
that these fructooligosaccharides were degraded into shorter
oligosaccharides
by liberating fructose from their fructosyl terminal.
This enzyme
can also hydrolyze
-2,1-linkages of sucrose (Table
1 and
Fig.
5), although the reaction seemed to be slow. These data led to
the
conclusion that this enzyme is capable of hydrolyzing not
only
-2,6-fructosyl linkages, but also
-2,1-fructosyl linkages.
It was
generally indicated that a bacterial levan had many branching
points
linked by
-2,1-fructosyl linkages at the C-1 position
of fructose
residues of its main
-2,6-linkage (
4). It can
be
considered that the exo-acting LDE reaction would be slowed
down or
terminated at these branching points. Therefore, the results
presented
above indicate that the enzyme from
S. exfoliatus F3-2
might
have degradation activity for
-2,1-fructosyl linkages.
Thus, the
enzyme is expected to have the potential for high-yield
production of
levanbiose from bacterial levan not only from
S. levanicum
(
5), but also from other microorganisms.
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FIG. 5.
TLC analysis of the reaction products from
fructooligosaccharides with purified LDE. The enzyme reaction was done
as described in Materials and Methods. G/F, glucose/fructose; GF,
sucrose; GF2, 1-kestose; GF3, nystose;
GF4, 1-fructosylnystose.
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To date, there have been few reports on levanbiose-producing LDEs
(
2,
8-10). Table
2 summarizes
the properties of the
known levanbiose-producing LDEs together with
those from
S. exfoliatus F3-2. As can be seen from the
table, the enzyme from strain F3-2
differs from the other enzymes in
terms of its range of stability
(higher stability under acidic
conditions), mode of action, and
effect on
-2,1-fructosyl linkages.
Therefore, the enzyme from
S. exfoliatus F3-2 was judged to
be a novel levanbiose-producing
LDE that can be clearly classified as
2,6-
-
D-fructan 6-levanbiohydrolase
(EC 3.2.1.64). It can
also be said that this is the first example
of purification of the
typical 2,6-
-
D-fructan 6-levanbiohydrolase.
These
results obtained with the enzyme of
S. exfoliatus F3-2 would
be important for providing enzymological characteristics of LDEs,
especially that of 2,6-
-
D-fructan 6-levanbiohydrolase.
To obtain
molecular information about this LDE and to prepare it more
effectively
for levanbiose production, cloning of the enzyme is in
progress.
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TABLE 2.
Properties of purified 2,6--D-fructan
6-levanbiohydrolase from S. exfoliatus F3-2 and of the other
levanbiose-producing LDEs
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ACKNOWLEDGMENTS |
We are grateful to Meiji Seika Kaisya, Ltd., for kindly supplying fructooligosaccharides.
This work was supported in part by a Grant-in-Aid for Scientific
Research (no. 07556087) from the Ministry of Education, Science, Sports
and Culture of Japan and also by a Grant-in-Aid for JSPS Fellows (no.
11-5613) to K.S. from the Japan Society for the Promotion of Science.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Microbial Resources and Ecology, Research Group of Molecular
Bioscience, Division of Applied Bioscience, Graduate School of
Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo
060-8589, Japan. Phone: (81)-11-706-2501. Fax: (81)-11-706-4961.
E-mail: yokota{at}chem.agr.hokudai.ac.jp.
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Applied and Environmental Microbiology, January 2000, p. 252-256, Vol. 66, No. 1
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Abstract
Introduction
