FEMS Microbiology Letters 55 (1988) 147-150
Published by Elsevier
147
FEM 03292
a-Glucose-l-phosphate formation
by a novel trehalose phosphorylase from Flammulina velutipes
Yutaka K i t a m o t o 1, Hajime Akashi
1,2, Hisashi T a n a k a
1 and N o b u h i r o Mori 1
Department of Agricultural Chemistry, Tottori University Koyama, Tottori, Japan
and e Research Center, Nisshin Flour Milling, Co., Ltd., Ohi-machi, Iruma-gun, Saitama, Japan
Received 10 May 1988
Accepted 1 June 1988
Key words: a-Glucose-l-phosphate; Trehalose phosphorylase; Flammulina velutipes
1. SUMMARY
A novel type of trehalose phosphorylase was
found in a basidiomycete, Flammulina velutipes.
The enzyme catalyzes both the reversible phosphorolysis of trehalose to form a-glucose 1-phosphate and glucose and also the synthesis of trehalose. Comparison of the specific activity of trehalose phosphorylase with that of trehalase suggested that the function of the former enzyme was
more important in the fruit-bodies of this fungus.
2. INTRODUCTION
The mycelia and fruit-bodies of a basidiomycete, Flammulina velutipes, contain much trehalose
[1]. In many microorganisms, trehalose is hydrolyzed to produce two glucose molecules by trehalase [2], but the phosphorolytic cleavage of trehalose into fl-glucose 1-phosphate (fl-Glc l-P) and
glucose by a phosphorylase has been reported for
Euglena gracilis [3]. In F. velutipes (as described
later), the trehalase activity was higher in the
mycelia than in the fruit-bodies, although a-Glc
Correspondence to: Y. Kitamoto, Department of Agricultural
Chemistry, Tottori University Koyama, Tottori 680, Japan.
1-P formation by trehalose phosphorylase was not
be detected. We found a novel trehalose phosphorylase in F. velutipes. This enzyme catalyzes
the following reversible reaction:
Trehalose + Pi ~ a-Glc 1-P + Glucose
3. MATERIALS AND METHODS
3.1. Culturing and collection of material
F. velutipes, stock 721-B1, was used for all
experiments. It was grown on potato-glucose liquid
medium as described by Kitamoto and Gruen [1].
Vegetative mycelia were collected 14 days after
inoculation when the colony had not fruited; at 26
days, the fruit-bodies that had arisen and the
mycelia of the developing colony were separated
and harvested. Fruit-bodies and mycelia were
washed thoroughly with distilled water and stored
in a deep freezer.
3.2. Partial purification of trehalose phosphorylase
Crude extracts were prepared by homogenization of the fruit-bodies in 100 mM potassium
phosphate buffer containing 25% glycerol and 5
mM EDTA (pH 7.0). After removal of cell debris
by centrifugation, the supernatant was fractionated
by ammonium sulfate precipitation between 35
0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies
148
and 55% saturation. The enzyme was dialyzed
against the same buffer (40 mM) and then put on
a DEAE-cellulose column. The elution was done
with a linear gradient of 40 to 500 m M of the
phosphate buffer. The active fractions were concentrated with Centriflo CF-50 (Amicon Corp.,
U.S.A.) and used as the purified enzyme.
3.3. Enzyme assay
The reaction mixture for synthesis of trehalose
contained 100 mM MES (2-(N-morpholino)ethane
sulfonic acid) and 100 m M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH
6.3), 1 M glucose, 100 mM a-Glc l-P, 0.1 mg
bovine serum albumin (BSA), and the enzyme in a
total volume of 1.0 ml. After incubation at 30 ° C
for 15 min, the Pi liberated was measured by the
purine nucleotide phosphorylase method of Machida and Nakanishi [4].
The reaction mixture for the phosphorolysis
contained 200 m M trehalose, 0.1 mg BSA, 40 m M
potassium phosphate buffer (pH 7.0), and the
enzyme in a total volume of 1.0 ml. After incubation of the same mixture at 30 ° C for 15 rain, the
glucose liberated was measured by the method of
Somogyi [5]. The activity was also assayed spectrophotometrically. The assay mixture contained
40 mM potassium phosphate buffer (pH 7.0), 200
mM trehalose, 10 mM GSH, 1.3 mM MgC12,
0.067 mM a-Glc 1,6-diphosphate, 1.0 m M NADP,
0.16 mM EDTA, 3 units of a-phosphoglucomutase, 3 units of glucose 6-phosphate dehydrogenase, and the enzyme, in a total volume of 3.0
ml. The increase in absorbance at 340 nm was
monitored at 30 ° C with a spectrophotometer.
Trehalase was assayed by the method of Kalf
and Rieder [6]. The glucose liberated was measured by the colorimetric method described above.
One unit of enzyme activity is defined as the
amount of enzyme needed for the formation of 1
/zmol of product per rain. Protein was assayed by
the method of Lowry et al. [7].
4. RESULTS A N D DISCUSSION
A product of the phosphorolysis of trehalose
was isolated. A preparative mixture (2.0 ml) was
Table 1
Paper chromatography of the product of enzyme action and
authentic a-Glc 1P
Substance
Enzymaticproduct
Authentic a-Glc 1-P
Rf value
Solvent l
0.27
0.28
Solvent 2
0.64
0.63
The analytical runs were carried out on Toyo No. 52 paper.
Phosphorus spots were made visible by the method of Hanes
and Isherwood [10]. Solvent 1, n-butanol/acetic acid/water
(2 : l : 1, v/v); solvent 2, ethanol/1 M ammonium acetate, pH
3.8 (7 : 3, v/v).
composed of 200 m M trehalose, 0.5 mg BSA, 40
m M potassium phosphate buffer (pH 7.0), and the
purified enzyme. The reaction was carried out at
3 0 ° C for 4 h by feeding of 5 /~1 of the enzyme
(0.08 units) eight times with intervals of 30 min.
By this reaction, an amount (23.8/~mol) of a-Glc
1-P equimolar to that of glucose was formed. The
phosphoric ester formed was isolated by the
method of McCready and Hassid [8]. The potassium salt o f the product obtained showed the
same Rf value as that of authentic a-Glc 1-P
(Sigma Chemical Co., U.S.A.) by paper chromatography (Table 1). The sugar phosphate reacted with a-phosphoglucomutase (Sigma) from
rabbit muscle, which cannot react with/3-Glc l-P,
and the formation of Glc 6-P was detected in the
coupling reaction with glucose 6-phosphate dehydrogenase.
The sugar product of the synthetic enzyme
reaction was prepared and identified. The preparative reaction mixture was composed of 100
m M MES and 100 m M HEPES (pH 6.3), 400 mM
glucose, 100 mM a-Glc l-P, 0.5 mg BSA, and the
purified enzyme, in a total volume of 1.0 ml. The
reaction was carried out by feeding of 10/H of the
enzyme (0.15 units) repeatedly as described above.
By this reaction, a total of 14 /~mol of Pi was
liberated, The enzymatic sugar product was isolated from the substrate by use of a charcoal
column as described by Marechal and Belocopitow [3]. The trimethylsilyl derivative of the product was prepared by the procedure of Sweeley et
al. [10], and analyzed by a temperature-programmed gas chromatograph equipped with a SE-
149
Table 2
Trehalase and trehalose phosphorylase activities in the
mycelium and fruit-bodies during development of F velutipes
Colony
age
(days)
Colony
portion
TH activity 1
(units/mg
protein)
TP activity 2
(units/mg
protein)
14
26
Mycelia
Mycelia
Fruit-bodies
0.017
0.023
0.003
0.046
0.019
0.036
about 26% of the trehalose phosphorylase level.
However, trehalase activity in the fruit-bodies at
26 days was only a b o u t 13% that in the mycelia of
the same colony. T h e specific activity of phosphorylase was a b o u t 12-fold that of the hydrolyase
in the fruit-bodies (Table 2). Trehalose phosphorylase in the fruit-bodies m a y act in the degradation of trehalose translocated from the
mycelia.
i TH, trehalase, 2 TP, trehalose phosphorylase. TP activity was
measured by the spectrophotometric method.
REFERENCES
30 column. The retention time of the resulting
trimethylsilyl derivative coincided with that for
authentic a,a-trehalose (Aldrich, U.S.A.).
The enzyme was specific toward trehalose, but
inert toward cellobiose, laminaribiose, maltose,
sucrose, glycogen, laminarin, and starch. The apparent K m values for trehalose and Pi in the
phospholytic reaction at the o p t i m u m pH, 7.0,
were 75 m M and 5.0 raM, respectively. Those for
glucose and ct-Glc 1-P in the synthetic reaction at
p H 6.3 were 0.63 M and 47 raM, respectively. It
seems that the enzyme was active in phosphorolysis only u n d e r physiological conditions.
Trehalose seems to be the major translocation
c a r b o h y d r a t e f r o m mycelia into fruit-bodies in F.
velutipes [1]. The specific activity of trehalase in
cell extracts f r o m the mycelia at 14 days was
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