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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 7, Num. 3, 2004, pp. 277-284
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Electronic Journal of Biotechnology, Vol. 7, No.3, December, 2004, pp. 274-281
RESEARCH ARTICLE
Isolation,
purification, and characterization of L-glutamate oxidase from Streptomyces sp.
18G
Supawadee Wachiratianchai1, Amaret
Bhumiratana2, Suchat Udomsopagit*3
1Department
of Biotechnology
Faculty of Science, Mahidol University
Rama 6 Road, Bangkok 10400 Thailand
Tel: 662 2015310
Fax: 662 2463026
E-mail: g4236968@student.mahidol.ac.th
2Department
of Biotechnology
Faculty of Science, Mahidol University
Rama 6 Road, Bangkok 10400 Thailand
Tel: 662 2015310
Fax: 662 2463026
E-mail: scabr@mahidol.ac.th
3National Center for
Biotechnology and Genetic Engineering
National Science and Technology Development Agency
113 Phaholyothin Road,
Klong 1, Klong Luang
Rangsit, Pathumthani 12120, Thailand
Tel: 662 5646700 ext 3423
Fax: 662 5646701-5
E-mail: suchat@biotec.or.th
*Corresponding
author
Financial support: Grant
PDF/15/2542 from Thailand Research Fund.
Code Number: ej04031
ABSTRACT
An extracellular L-glutamate
oxidase (GLOD) was purified from soil-isolated Streptomyces sp 18G.
The enzyme had a molecular weight of approximately 120,000 and consisted
of two identical subunits, each with a molecular weight of 61,000. The
isoelectric point was pH 8.5 and the enzyme had an optimal pH between 7.07.4.
GLOD showed the maximum activity at 37ºC.
The GLOD activity was stable at pH ranging from 6.5 to 7.0 for 1 hr. Among
21 amino acids tested for substrate specificity, L-glutamate was almost
exclusively oxidized. D-glutamate and L-aspartate were oxidized but only
to extents of 0.79% and 0.53%, respectively.
Keywords:L-glutamate, L-glutamate oxidase,
purification, screening, Streptomyces.
Abbreviations:
GDC: Glutamate decarboxylase
GDH: Glutamate dehydrogenase;
GLOD: L-glutamate oxidase.
ARTICLE
L-glutamate
is an important amino acid widely used as a food additive because of its
taste enhancing property. In neurochemistry, it is a major excitatory neurotransmitter
of the central nervous system and the enteric nervous system (Cooper and
Pritchard, 1994; Zilkha et al. 1995; Valero and García-Carmona, 1998). Based
on this information, it is obviously very important to develop specific analytical
methods for measuring this amino acid, preferably in simple and reliable
way (Valero et al. 1998).
L-glutamate
can be measured by chromatographic methods (Kondrat et al. 2002; Hanko and
Rohrer, 2004) which are complicated, time-consuming and require extensive
sample pretreatment. The enzymatic method is chosen to overcome the problems
mentioned above. Glutamate dehydrogenase (GDH) and glutamate decarboxylase
(GDC) have been employed for the determination of L-glutamate (Shi and Stein,
1996; Liu et al. 1999; Ling, et al. 2000; Oliveira et al. 2001; Qhobosheane
et al. 2004; Rodriguez et al. 2004). However, the GDC and GDH have some drawbacks
due to poor substrate specificity and the requirement for expensive coenzyme
such as NAD+. L-glutamate oxidase (GLOD) is used instead due to
the relatively high substrate specificity comparing to GDH and GDC and no
requirement for additional coenzyme. GLOD is an enzyme that specifically
catalyzes the oxidative deamination of L-glutamate in the presence of water
and oxygen with the formation of a-ketoglutarate, ammonia and hydrogen peroxide
(Kusakabe et al. 1983; Böhmer et al. 1989; Fukunaga et al. 1998). The hydrogen
peroxide formed in this reaction can easily be detected by the chromogenic
peroxidase reaction or amperometric method (Böhmer et al. 1989; Villarta
et al. 1991; Almeida and Mulchandani, 1993; Zilkha et al. 1995; Niwa et al.
1997; Chang et al. 2003). Therefore, L-glutamate oxidase holds excellent
potential for use as the principle component in the determination of L-glutamate
(Chen and Su, 1991; White et al. 1994; Ye et al. 1995; Matsumoto et al. 1998;
Udomsopagit et al. 1998; Valero and Garcia-Carmona, 1998; Yao et al. 1998),
although the presently available GLOD still has several disadvantages such
as broad substrate specificity of the enzyme from some microorganisms (Kamei
et al. 1983) and high cost (Kusakabe et al. 1983). In this
study, we conducted a screening for glutamate oxidase-producing microorganisms
from natural sources and investigating the physical and biochemical characteristics
of the GLOD after the purification steps.
MATERIALS AND METHODS
Materials
SP-Sepharose
Fast Flow, Q-Sepharose Fast Flow, and Superdex 200 HR 10/30 were from Amershame
Biosciences Ltd. (Uppsala, Sweden).
Protein markers for gel filtration and amino acids were from Sigma-Aldrich
Co. (St. Louis, USA).
Microorganisms
and culture conditions
Streptomyces sp.
18G was isolated from soil sample in Khlong Luang District, Pathum
Thani Province, Thailand.
The medium used for the screening was humic acid-vitamin agar (Hayakawa and
Nonomura, 1987). The microorganism grown on inorganic salt starch agar medium
(Williams et al.1983) had spiral mycelia with spores. The colonies were tough,
leathery and developed to powder and velvet colonies after spore forming.
These characteristics suggested that the GLOD producingmicroorganism was
of the genus Streptomyces.
Screening
methods
The GLOD
producing strain was selected by using the method based on H2O2-dependent
peroxidase catalyzed chromogenic reaction, as described by Li et al. 1996.
Filter papers were dipped into the reaction mixture containing 2 U/ml horseradish
peroxidase (HRP), 10 mmole monosodium glutamate (MSG), 10 mmole 4aminoantipyrine
(AAP) and 17.5 mmole phenol in 0.1 M potassium phosphate buffer pH 7.4,
prior to placing on the isolated colonies. The reaction was allowed to take
place by incubating for 15-30 minutes at ambient temperature without light.
GLOD producing colonies that turned the filter paper red were collected.
GLOD
production culture
Streptomyces sp.
18G was grown in wheat bran precultivation medium described by Böhmer et
al. 1989 with some modifications. The medium contained 2.0% wheat
bran, 0.5% sodium chloride and 0.5% monosodium glutamate. The culture was
grown at 37ºC with shaking at 200 rpm.
GLOD
assay
GLOD activity
was assayed following the methods described by Li et al. 1996. The reaction
mixture contained 1.0 mmole 4aminoantipyrine, 17.5 mmole phenol, 2.5 U horseradish
peroxidase and a sufficient amount of GLOD in 0.1 M potassium phosphate buffer pH 7.4
in a total volume of 1.30 ml. After a pre-incubation
for 2 min at 37ºC, the reaction was initiated
by addition of 10 mmole MSG to the reaction mixture. The absorbance at 500
nm was measured after incubated for 30 min at 37ºC with
gentle shaking. One unit of enzyme activity is defined as the amount of enzyme
required to produce 1 mmol of H2O2 per minute under
the assay conditions.
Protein
determination
Protein
was determined using Bradford assay. Bovine serum
albumin (BSA) was used as a standard.
Purification
of GLOD
Enzyme
concentration. Ammonium sulphate precipitation technique was used to
concentrate the crude enzyme. Grounded ammonium sulphate was gradually
added to the chilled enzyme solution while stirring until 80% saturation
was obtained. The solution was then stirred at 0ºC for 1 hr The precipitate was
collected by centrifugation at 10,000 rpm, at 4ºC for 45 min. The protein pellet
was dissolved in a minimal volume of 20 mM chilled potassium phosphate buffer
pH 6.0. The enzyme solution was dialyzed overnight against the same buffer
at 4ºC.
Cation-exchange
chromatography. The concentrated enzyme was applied to SP-Sepharose
FF column with a bed volume of 30 ml. The column was pre-equilibrated with 20 mM potassium phosphate buffer pH 6.0.
After the column was washed with 40 ml of potassium phosphate buffer, the
enzyme was eluted with a linear salt gradient of 0-.3 M sodium chloride at a flow rate of
1 ml/min. Each fraction of the enzyme solution was tested for GLOD activity.
The active fractions were pooled and concentrated by means of a centrifugal
ultrafiltration and kept in an ice bath for further purification steps.
Anion-exchange
chromatography. The pooled active fractions were desalted prior to
loading into the pre-equilibrated Q-Sepharose Fast Flow column with a bed
volume of 30 ml. The column was washed with 20
mM Tris-HCl buffer pH 8.0 at the flow rate of 1 ml/min.
Other proteins which bound to the column were then eluted by using linear
salt gradient from 0.0-1.0 M sodium chloride at the same flow
rate. The active fractions were pooled and concentrated by means of the
centrifugal ultrafiltration with MW cut off at 10,000 and kept in an ice
bath for the next step of enzyme purification.
Gel
filtration chromatography. Gel filtration chromatography was used for
the last step of enzyme purification. The Superdex 200 HR 10/30 column
with 24 ml bed volume was pre-equilibrated with 50
mM sodium phosphate buffer pH 7.0 with 0.15
M sodium chloride. The concentrated enzyme from the
previous step was applied. The enzyme was eluted from the column with the
same buffer at a flow rate of 0.5 ml/min. Active fractions were determined
for GLOD activity by the colorimetric method, and were pooled and concentrated
for further assays.
SDS-polyacrylamide
gel electrophoresis
SDS-PAGE
slab gel was carried out using a Mini-PROTEAN3® cell (Bio-Rad,
Hercules, USA).
Molecular weight markers were obtained from Sigma-Aldrich
Corp., St. Louis, USA.
The gels were subjected to protein bands visualization with silver staining
method.
Molecular
weight determination by gel filtration chromatography
The relative
molecular weight (Mr) of the native enzyme was determined by using
Superdex 200 HR 10/30 column. Elution was done at the flow rate of 0.25 ml/min
with an elution buffer comprising 50 mM sodium phosphate buffer pH 7.0
and 0.15 M NaCl.
The calibration curve was constructed using protein markers: cytochrome C
(12,400), carbonic anhydrase (29,000), bovine serum albumin (66,000), alcohol
dehydrogenase (150,000) and b-amylase (200,000). Dextran blue (2,000,000)
and vitamin B12 (1,355.4) were used to determine the void volume
(Vo) and total volume (Vt), respectively. A calibration
curve between log molecular weights of protein markers and the partition
coefficient values, Kav, was constructed.
Isoelectric
point estimation
The isoelectric
point was determined by using PhastGelâ IEF 3-9 (Amersham
Biosciences, Uppsala, Sweden).
The standard pI markers consisted of amyloglucosidase (3.5), soybean
trypsin inhibitor (4.55), b-lactoglobulin A (5.20), bovine carbonic anhydrase
B (5.85), human carbonic anhydrase B (6.55), myoglobin-acidic band (6.85),
myoglobin-basic band (7.35), lentil lectin-acidic band (8.15), lentil lectin-middle
band (8.45), lentil lectin-base band (8.65) and trypsinogen (9.30). Plots
of the distances of the protein markers from the anode and their pIs
were constructed and fitted using linear regression. The pI of the
GLOD was estimated using a regression equation.
RESULTS
Enzyme
production
The extracellular
GLOD was produced in wheat bran precultivation medium (Böhmer et al. 1989).
GLOD activity obtained was 13.79 mU/ml. The composition of the medium was
modified to formulate the medium that promoted the higher GLOD production.
The effect of MSG added in the medium to the GLOD productivity was also investigated.
There was no improvement in the enzyme productivity by adding glucose as
a carbon source. The optimal GLOD production medium containing 2.0% wheat
bran, 0.5% NaCl and 0.5% MSG was selected and named wheat bran medium. The
maximum GLOD production was obtained from the cultivation of Streptomyces sp.
18G in wheat bran medium at 30ºC for
60 hrs with shaking at 200 rpm.
Purification
of GLOD
GLOD was
purified from an extract of Streptomyces sp. 18G cultured on wheat
bran medium. The procedure included precipitation with ammonium sulphate,
column chromatography on SP-SepharoseFF, Q-SepharoseFF and a high resolution
gel filtration on Superdex 200 HR 10/30 as described above. Table
1 summarizes the purification of the enzyme. The overall purification
was 990 fold with a yield of 16.65%. The purified enzyme showed a single
band in SDS-PAGE and had a specific activity of 152.35 U mg-1 (Figure
1).
Table
1. Purification of GLOD from Streptomyces sp. 18G.
|
Purification
step
|
Total
protein (mg)
|
Total
activity (U)
|
Specific
activity
(U
mg-1)
|
Yield
(%)
|
Purification
(fold)
|
Culture extract
Ammonium sulphate
SP-SepharoseFF
Q -SepharoseFF
Gel filtration
|
1,203
376.6
10.7
0.72
0.2
|
185.25
151.03
70.23
33.43
30.85
|
0.15
0.4
6.56
46.48
152.36
|
100
81.53
37.91
18.05
16.65
|
1.0
2.6
42.7
302
990
|
Molecular
weight and subunit structure
The relative
molecular weight (Mr) of the native enzyme was estimated to be
approximately 120,000 by Superdex 200 HR 10/30 gel filtration chromatography
(Figure 2). The subunit structure of the enzyme was
analyzed by SDS-PAGE in 3 different polyacrylamide separating gel concentrations, i.e. 10%,
12% and 14% using a 4% stacking gel. Molecular weight of the enzyme subunits
calculated from the three regression equations at different gel concentrations
were 59,816, 63,252 and 60,044, respectively (Figure 3).
The molecular weight of the GLOD subunit was estimated to be 61,000. Since
the native enzyme was approximately twice the size of the enzyme subunit,
the results suggested that the enzyme consisted of two identical subunits.
Isoelectric
point
The isoelectric
point of the purified GLOD was estimated to be 8.5 by isoelectric focusing
(Figure 4).
Optimal
pH and pH stability
Figure
5A shows the pH-activity profile of GLOD. The enzyme showed maximum
activity in the pH range from 7.0 to 7.4. The enzyme was more stable in
alkaline pH than in acidic pH (Figure 5B).
Optimal
temperature and thermal stability
As illustrated
in Figure 6A, GLOD activity showed maximum activity
at 37ºC under
standard assay conditions described above. Thermal stability of GLOD was
determined by incubating the enzyme in 0.1
M potassium phosphate buffer pH 7.4 at various temperatures
for 1 hr. The enzyme was relatively stable from 30 to 55ºC. At 65ºC, the enzyme showed approximately
50% of the original activity. The enzyme was completely inactivated at 75ºC.
Substrate
specificity
The activity
of GLOD on various amino acids was investigated. Table
2 illustrates that L-glutamate was almost exclusively oxidized. In addition
to L-glutamate, D-glutamate and L-aspartate were oxidized but with relative
activities of 0.79% and 0.53%, respectively. The activities on other amino
acids tested were undetectable.
Table
2. Substrate
specificity of GLOD. The enzyme activity was measured as described
in text. All values represent the percentage activity compared with
the value obtained from L-glutamate. UD = undetectable.
|
Substrate
(10 mM)
|
Relative
activity (%)
|
L-glutamate
|
100
|
D-glutamate
|
0.79
|
L-glutamine
|
UD
|
L-aspartate
|
0.53
|
L-glycine
|
UD
|
L-alanine
|
UD
|
L-arginine
|
UD
|
L-cysteine
|
UD
|
L-histidine
|
UD
|
trans-4-hydroxy-L-proline
|
UD
|
L-lysine
hydrochloride
|
UD
|
L-proline
|
UD
|
L-serine
|
UD
|
L-threonine
|
UD
|
L-valine
|
UD
|
L-isoleucine
|
UD
|
L-methionine
|
UD
|
L-phenylalanine
|
UD
|
L-leucine
|
UD
|
L-tryptophan
|
UD
|
L-tyrosine (1
mM)
|
UD
|
DISCUSSION
An extracellular
GLOD was isolated from Streptomyces sp. 18G. The enzyme was efficiently
produced in wheat bran medium. The enzyme was purified approximately 990-fold
from the culture broth with 16.65% yield. The specific activity was 152.36
U mg-1. The enzyme obtained in this study is different from those
reported in previous studies in several aspects. The native enzyme has a
molecular weight of approximately 120,000 Da with 2 identical subunits. The
results were in accordance with previous studies by Böhmer et al. 1989 and
Patel et al. 2000. However, Kusakabe et al.1983 reported that GLOD from Streptomyces sp.
X-119-6 had a molecular weight of approximately 140 kDa and consisted of
3 types of subunits, a, b and g with molecular weights of approximately 44,
19 and 9 kDa, respectively. Recently, another GLOD with three subunits was
obtained from S. platensis NTU3304 (Chen et al. 2001). The pI of
GLOD from Streptomyces sp. 18G was estimated to be 8.5 which differed
from those obtained from S. endus and Streptomyces sp. X-119-6
with pI 6.2 in previous studies (Kusakabe et
al. 1983; Böhmer et al. 1989). The pH-activity and temperature-activity profiles
suggested that the optimal pH ranged from 7.0 to 7.4 and the optimal temperature
was 37ºC,
which is applicable for glutamate determination under physiological conditions.
In addition, the enzyme shows almost exclusively oxidize L-glutamate with
lower extent on other amino acids tested. However, a low GLOD production
was obtained from Streptomyces sp. 18G in this study. This result
implies that control of the culture conditions, i.e. pH, salts, etc.
may facilitate GLOD production. Arima and colleagues (2003) were successful
in cloning and expressing the gene encoded for the enzyme in Escherichia
coli. The report showed that proteolysis of the enzyme by metalloendopeptidase
from Streptomyces griseus (Sgmp) could stabilize the recombinant enzyme
and improvedits catalytic efficiency at various pH. The report suggests that
the DNA recombination technology can improve the GLOD production and stability.
Based
on the results of this study, the GLOD from Streptomyces sp. 18G may
have a potential for development of analytical systems for the specific determination
of L-glutamate such as biosensors or kits for clinical diagnosis, bioprocess
monitoring and food quality control. Additional studies are needed to obtain
deeper insight into catalytic and physiochemical properties of the enzyme.
Besides, molecular biology and bioprocess control may help promoting the
production and stabilization of the enzyme.
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