2220
S. ENOKIBARA
(Wakenyaku, Japan) at 5 ꢀC for 3 min for cell disruption. Cell debris
was removed by centrifugation, and the supernatant was assayed for
choline oxidase activity. The degree of cell growth in the choline liquid
medium, the productivity, and the pH requirements of the enzyme were
examined.
equilibrated using the buffer. The column was washed with the
equilibrated buffer, and the enzyme was eluted with a linear gradient of
NaCl (0–0.5 M) in the buffer. The active fractions were pooled and
concentrated by the addition of ammonium sulfate to 80% saturation.
The precipitate formed was collected by centrifugation and dissolved
in a minimal volume of the buffer.
Identification. Identification of the isolated strain was done by the
National Collections of Industrial, Food and Marine Bacteria (NCIMB
Japan, Shizuoka). The morphological characteristics and the 28S
rDNA-D1/D2 sequence of strain V2 were investigated. A homology
search for the 28S rDNA-D1/D2 sequence was performed by
BLAST.17)
Sephacryl S-200 column chromatography (1st). The enzyme solu-
tion was applied to
a
Sephacryl S-200 column (1:4 ꢂ 76 cm)
equilibrated with the buffer, and was eluted with the buffer. Active
fractions were combined and concentrated by the addition of
ammonium sulfate to 80% saturation. The precipitate formed was
collected by centrifugation and dissolved in 10 mM potassium
phosphate buffer (pH 8.0) containing 0.1 mM DTT. The enzyme
solution was dialyzed against the buffer.
Organism and cultivation. Strain V2 isolated from soil was used.
The culture medium contained 1% choline chloride, 0.3% K2HPO4,
.
0.3% KH2PO4, 0.05% yeast extract, and 0.02% MgSO4 7H2O at
pH 8.0. Cultivation was carried out at 30 ꢀC for 5 d in a 500 mL
shaking flask containing 100 mL of the medium, with reciprocal
shaking.
Hydroxyapatite column chromatography. The dialyzed solution was
applied to a hydroxyapatite column (1:4 ꢂ 3:2 cm) equilibrated with
10 mM potassium phosphate buffer (pH 8.0) containing 0.1 mM DTT.
The column was washed with the equilibrated buffer, and the enzyme
was eluted using a linear gradient of phosphate buffer (0.01–0.2 M).
Active fractions were combined and concentrated by the addition of
ammonium sulfate to 80% saturation. The precipitate obtained was
dissolved in a minimal volume of the buffer.
Enzyme assay. Choline oxidase activity was assayed by the quantity
of betaine aldehyde or H2O2 formed. For estimation of the formation
of betaine aldehyde, the reaction mixture contained 90 mmol of
glycine-NaOH buffer (pH 9.5), 10 mmol of choline chloride, and the
enzyme in a total volume of 1 mL. The reaction was carried out with
shaking at 30 ꢀC for 10 min. The quantity of betaine aldehyde in the
reaction mixture was determined by a modification of a procedure
described by Jellinek et al.,18) as follows: One mL of the reaction
mixture was mixed with 0.1% 2,4-dinitrophenylhydrazine in 1 M HCl
and heated in a boiling water bath for 7 min. The mixture was cooled to
room temperature, and then 6.4 mL of water was added. NaOH (2 M,
1.6 mL) was added, and then the absorbance at 440 nm was measured.
To measure the formation of H2O2, a method involving peroxidase,
phenol, and 4-aminoantipyrine was used.19) The reaction mixture
contained 60 mmol of glycine-NaOH buffer (pH 9.5), 3 mmol of phenol,
2.25 mmol of 4-aminoantipyrine, 5 U of peroxidase, 15 mmol of choline
chloride, and a suitable amount of the enzyme in a total volume of
1.5 mL. The reaction was carried out at 30 ꢀC, and the increase in
Sephacryl S-200 column chromatography (2nd). The enzyme
solution was applied to the column as in step 4, and was eluted with
the buffer as in step 4. Fractions with enzyme activity were stored at
ꢁ80 ꢀC.
Polyacrylamide gel electrophoresis. Polyacrylamide gel electro-
phoresis (PAGE) was performed on commercially available gel plates
(Multi Gel II mini 7.5 for native-PAGE and Multi Gel II mini 10/20
for SDS–PAGE). The gels were stained with Coomassie Brilliant Blue
R-250.
Determination of molecular weight. The molecular weight of the
choline oxidase was estimated by gel filtration on a Sepharose 6B
column equilibrated with 0.05 M Tris–HCl buffer, pH 8.0, containing
0.1 mM DTT. The standard proteins used were thyroglobulin
(669 kDa), ferritin (450 kDa), catalase (240 kDa), bovine albumin
(68 kDa), chymotrypsinogen A (25 kDa), and cytochrome c (12.5 kDa).
absorbance at 505 nm was measured using
a Hitachi U-2000A
spectrophotometer (Hitachi, Tokyo). Enzyme activity was calculated
using an extinction coefficient of 9.2 mMꢁ1 cmꢁ1 for quinine-imine dye
at 505 nm, pH 9.5. One unit of enzyme activity was defined as the
quantity of enzyme necessary to catalyze the formation of 1 mmol of
betaine aldehyde or H2O2 per min. Choline oxidase activity was
estimated by measurement of the H2O2 formed in the reaction mixture,
unless otherwise stated.
Results and Discussion
Protein determination. Protein levels were determined by Bio-Rad
Protein Assay (Hercules, CA) with bovine serum albumin as standard,
or by the absorbance at 280 nm.
Screening and identification of the microorganism
A total of 244 microorganisms that were grew on
choline as sole carbon and nitrogen source at pH 10
were isolated from 50 Japanese soil samples. Among
these isolates, three strains of filamentous fungi showed
significantly faster and more profuse growth on choline
liquid medium at pH 10. A fungus designated strain V2
exhibited the highest specific choline oxidase activity
and highest productivity in the cell-free extract. Hence it
was selected for the present study. It was identified by
NCIMB Japan as F. oxysporum in that its 28S rDNA-
D1/D2 sequence showed more than 99% similarity to
that species.
Purification of choline oxidase. Purification of choline oxidase from
strain V2 was carried out at 5 ꢀC. Tris–HCl buffer (50 mM, pH 8.0)
containing 0.1 mM dithiothreitol (DTT) was used, and centrifugation
was performed at 40;000 g for 20 min throughout the enzyme
purification procedure, unless otherwise stated.
Preparation of cell-free extract. The washed cells, harvested from
4 L of culture medium, were suspended in 1 L of Tris–HCl buffer and
disrupted for 90 min using an ultrasonic oscillator (19 kHz) on ice. The
disrupted cells were centrifuged, and the supernatant was used as cell-
free extract.
Choline oxidase production by strain V2
Ammonium sulfate fractionation. During this procedure, the pH of
the solution was kept at about 8.0 with 14% ammonium hydroxide
solution. Ammonium sulfate was added to the cell-free extract to 50%
saturation and the precipitate was discarded. A precipitate was formed
by further addition of ammonium sulfate to 80% saturation, and was
collected by centrifugation. The precipitate recovered was dissolved in
Tris–HCl buffer and dialyzed against the buffer (2 L) for 15 h.
The production of choline oxidase by strain V2
was examined under the conditions described above.
Strain V2 showed better growth at pH 8.0 than at
pH 10.2 when a large culture was grown in a 2 L
shaking flask containing 300 mL of medium, with
reciprocal shaking (data not shown). Choline oxidase
activity increased with cell growth, reaching a maximum
at 4–5 d.
DEAE-Toyopearl column chromatography. The dialyzed enzyme
solution was applied to a DEAE-Toyopearl column (2:4 ꢂ 26:5 cm)