Determination of L-Glutaminase Activity by Some Bacterial Species

Full text

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
218
Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.504.027
Determination of L-Glutaminase Activity by Some Bacterial Species
Gulbahar F. Karim1 and Karkaz M. Thalij2*
1College of Nursing–University of Kirkuk-Iraq
2Department of Food Sciences-University of Tikrit-Iraq
*Corresponding author
A B S T R A C T
Introduction
Microbial enzymes play a major role in the
diagnosis, curing, biochemical investigation,
and monitoring of many diseases.
Microorganisms represent an excellent
source of many therapeutic enzymes owing
to their broad biochemical diversity and
their susceptibility to genetic manipulation.
The manufacture of enzymes for use as
drugs is an important facet of today's
pharmaceutical industry (Saptarshi and Lele,
2011). Biomedical sciences accentuate the
involvement of the enzyme L-Glutaminase
and other amino acid depleting enzymes as a
therapeutic agents for the treatment of tumor
(Holcenberg, 1982). L-Glutaminase (L-
glutamine amidohydrolase E.C 3.5.1.2) is a
hydrolytic enzyme that deaminates L-
glutamine to glutamic acid and ammonia
(Roberts et al., 1970). Another application
of L-glutaminase in food flavoring
especially in the soy souse and related
industries of the orient.
With the development of biotechnology,
microbial glutaminase found newer
application in clinical analysis and even in
manufacture of metabolites. It uses in
biosensors for monitoring glutamine levels
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 5 Number 4 (2016) pp. 218-225
Journal homepage: http://www.ijcmas.com
Out of 200 clinical samples, 178(89%) bacterial isolates were recovered. Based on,
cultural, morphological, and biochemical testes, there were 87 (48.88%) isolates of
Gram positive cocci belong to the genus Staphylococcus, including, 63(35.39%)
and 24(13.48%) isolates of Staph aureus and Staph epidermidis respectively.
Whereas the 91(51.12%) remainder isolates were belong to the family
Enterobacteriaceae and distributed as 56(31.46%), 23(12.92%) and, 12(6.74%)
isolates of E. coli, Pseudomonas aeruginosa and Citrobacter diversus respectively.
All the bacterial isolates were screened for L-glutaminase enzyme activityusing
rapid plate assay. Twenty six (14.61%) isolates were found to be L-glutaminase
producers. The zone index was calculated for all L-glutaminase producing samples
which are ranged from (3.0-0.25). The maximum zone index was recorded by
Pseudomonas aerogenosa. The enzymatic activity were ranged from(18.5-
6.9)IU/ml. However the maximum activity was recorded for E. coli. No.7, Hence
this isolate was selected to produce large scale from the L-glutaminase enzyme for
further investigations.
Ke ywor ds
L-glutaminase,
Production,
E. coli and
Pseudomonas
aerogenosa.
Accepted:
15 March 2016
Available Online:
10 April 2016
Article Info

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
219
in mammalian and hybridoma cell cultures
without the need of separate measurement of
glutamic acid (Sabu et al., 2002). Many
microorganisms, such as bacteria, yeasts,
moulds and filamentous fungi, have been
reported to produce L-glutaminase.
This enzyme from microbial origin
supposed to be more stable than that from
animal and plant sources (Sajitha et al.,
2013). Commercial production of
glutaminase is carried out using submerged
fermentation (SmF) technique. Also solid
state fermentation (SSF) has emerged as a
promising technology for the development
of several bioprocesses which include the
production of industrial enzymes on a large
scale (Athira et al., 2014). The main
objective of this study is to investigate the
production and determined the L-
glutaminase activity by some clinical
bacterial species.
Materials and Methods
Sample Collection, Isolation, and
Identification of Isolates
Two hundred Samples are collected from in
and out patients with wound infections
admitted to the Sulaimani Teaching Hospital
during the period from March 2014 to
December 2014. The samples were collected
using disposable sterile swabs, they
transferred immediately to the laboratories
for culturing in Brain heart infusion broth,
on Blood agar, Nutrient agar and
MacConkey agar, then incubated at 37ºC for
24 hours. There were178 samples yield
positive growth. Colonies are purified and
used for identification tests. All bacterial
isolates were examined by biochemical tests
according to Bergey's manual of
determinative bacteriology (Holt et al.,
1994). The results were confirmed by
performing Vitek technique. The culture was
maintained on Nutrient agar medium slants.
Inoculated slants were grown in an incubator
at 37 ˚C for 24 hr. After that the slants were
stored at 4 ˚C in a refrigerator for short term
preservation and sub cultured every 15 days
in the abovementioned medium.
Qualitative production of L-glutaminase
Enzyme (Screening Test, Rapid Plate
Assay)
The minimal agar media (g/l of distilled
water) contains NaCl, 0.5; KCl, 0.5;
MgSO4.7H2O, 0.5; KH2PO4,
1;FeSO47H2O, 0.1; ZnSO47H2O, 1; L-
glutamine, 0.5: as nitrogen source, and
supplemented with 2.5% phenol red dye
(prepared in ethanol and the pH was
adjusted to 7.0). Control plate was
maintained without glutamine (instead
containing NaNO3 as nitrogen source).
After autoclaving, the prepared media were
inoculated with 24hr. old bacterial colonies
then incubated at 37 ºC for 24 hr. The pink
zone around bacterial colonies were
observed, and the zone index was calculated
according to (Gulati et al., 1997).
Zone index = Diameter of zone produced by
L-Glutaminase (mm)/ Diameter of bacterial
colony (mm).
Inoculum Preparation
The inoculum for all L-glutaminase
producing isolates were prepared in 250 ml
Erlenmeyer flasks containing 100 ml of
above medium at pH 7.0. The medium was
autoclaved at 121 ºC (15 lb) for 15 min.,
then inoculated with the bacterial isolate.
The inoculated flasks were kept on a shaker
at 150 rpm for 24 hrs, then used as an
inoculums.
Quantitative Production of L-glutaminase
Enzyme (Large Scale)
The L-Glutaminase production medium

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
220
(GPM) was prepared according to Suresh
Kumar, et.al. ( Suresh Kumar et al., 2013)
with slight modification. The medium
composed of (g/l of distilledwater):
Galactose 10.0, Yeast extract 10.0, L-
Glutamine 10.0, Magnesium sulphate 0.5,
KH2PO4 0.5, K2HPO4 0.5, NaCl 10. These
components were dissolved and the volume
was made up to 1L with D.W. then each 100
ml dispensed in 250 ml Erlenmeyer flasks,
autoclaved at121 ºC (15 lb) for 30 min., then
they were aseptically inoculated with 3% of
the prepared inoculum from Glutaminase
producing isolates and incubated at 37 ºC for
24 hrs. at 150 rpm in shaker incubator.
The bacterial cells are harvested in
refrigerated centrifuge at 8000 rpm for 20
min at 4 °C. The supernatant was used for
enzymatic assay, and the cells washed twice
with 0.02M phosphate buffer PH 8. Then the
cells were disrupted by ultra sonication
(Soniprep 150 sonicator) for 5 min.
(intermittent) under cold conditions (Scopes,
1987). The supernatant was the source of
crud enzyme and used for further enzymatic
assay procedures.
Determination of Enzyme Activity
L-Glutaminase was assayed according to
(Imada et al., 1973). The reaction mixture,
containing 0.5ml of an enzyme
preparation,0.5 ml of L-glutamine (0.04 M),
0.5 ml of phosphate buffer 0.1 M (pH 8.0),
and 0.5 ml of distilled water to a total
volume of 2ml solution was incubated at
37°C for 30 min. The reaction was stopped
by addition of 0.5 ml of 1.5 M Trichloro
acetic acid. Then to 3.7 ml of distilled water,
0.1 ml of the above mixture and 0.2 ml of
Nessler’s reagent were added and color
developed was read after keeping the
mixture at 20°C for 20 min at 450 nm in a
spectrophotometer. Enzyme and substrate
blanks were used as controls. One unit of L-
Glutaminase activity was defined as the
amount of enzyme that liberated 1μmol of
ammonia per one minute under optimal
assay conditions. Assays were done in
triplicate and the mean enzyme activity was
expressed as International unit per ml
(IU/ml).
Results and Discussion
Isolation and Identification of Bacteria
From 178wound samples which yielded
positive growth, there were 87 (48.88%)
isolates of Gram positive cocci belong to the
genus Staphylococcus, including,
63(35.39%) and 24(13.48%) isolates of
Staph. aureus and Staph epidermidis
respectively. Whereas the 91(51.12%)
remainder isolates were belong to the family
Enterobacteriaceae and distributed as
56(31.46%), 23(12.92%) and, 12(6.74%)
isolates of E. coli, Pseudo aerogenosa, and
Citrobacter diversus respectively as
revealed in Table 1. These results depended
on morphological characteristics of bacterial
isolates on cultural media and Gram staining
as well as to the results obtained from
conventional biochemical tests as
represented in Table 2. and Table 3. The
diagnosis of these bacterial species were
confirmed by performing Vitek technique.
Qualitative Estimation of L-glutaminase
Activity
All the bacterial isolates were submitted to
the screening test for producing L-
glutaminase enzyme which carried out by
rapid plate assay (Gulati et al., 1997).
Out of 178 (89%) screened isolates, 26
(14.61%) bacteria were able to form pink
zone in plates, Characteristics of L-
glutaminase producing bacteria. The
bacterial L-glutaminase hydrolysed L-

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
221
glutamine to glutamate and ammonia. The
acid base indicator dye phenol red converts
in to pink colour at basic PH.The zone index
was calculated for all L-glutaminase
producing samples which are ranged from
(3.0 -0.25) as presented in (Table 4).
The maximum zone index was recorded by
Pseudo. aeruginosa, whereas the minimum
zone index was for six isolate of E.coli.
Three isolates of Pseudo. aeruginosa had
zone index of 2.75, while zone index of 2
was recorded for four isolates of Staph
aureus, followed by zone index of 1.75 that
recorded for two isolates of Staph.
epidermidis.
The zone index of 1.5 was recorded for three
isolates of Staph aureus and two isolates of
Staph. epidermidis. Where as zone index of
1.25 was recorded for two isolates of
Citrobacter diversus and zone index of 1
was recorded for one isolate of Citrobacter
diversus. Ultimately the zone index of 0.5
was recorded for one isolates of E.coli.
Many researchers were investigated the
production of L-glutaminase from varies
microbial origins, including bacteria as
Staph. Aureus, Pseudo. aeruginosa (Soda et
al., 1972; Oshima et al., 1976; Rashmi et al.,
2012), E.coli (Pruisner et al., 1976), yeast
and filamentous fungi(Elshafei et al., 2014).
The production titer value of these enzymes
are influenced by microbial strains and
fermentation conditions (Iyer and Singhal,
2008).
Quantitative Estimation of L-glutaminase
Activity
All the 26 Positive isolates which were
screened for L-glutaminase in the above step
were further cultured in Glutaminase
producing media (GPM) containing L-
glutamine as a sole carbon and nitrogen
source. Quantitative estimating of L-
glutaminase activity by selective isolates
was carried out using Nesslerization process.
The enzymatic activity were ranged from
(18.5-6.9) as shown in table 4.6.
However the maximum activity was
recorded for E. coli. No.7 despite the narrow
zone index that produce by this bacteria in
the previous step, this finding might be
attributed to intracellular production of the
enzyme by this bacteria (Hartman, 1968).
Hence this isolate was selected to produce
large scale from the enzyme L-glutaminase
for further study.
Table.1 Distribution of Bacterial Isolates from Wounds Infections
Total
isolates
Citr.
diversus
Staph
epidermis
P.
aeruginosa
E. coli
Staph.
aureus
Source
of
isolates
178
12
24
23
56
63
Infected
Wounds
100
6.74
13.48
12.92
31.46
35.39
(%)

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
222
Table.2 Type of Tests for Staph aureus and Staph epidermidis Identifications
Staph epidermidis
Staph. aureus
Identification tests
+
+
Gram stain
_
-
Motile tests
+
+
Catalase test
_
-
Oxidase test
_
+
Mannitol salt fermentation
+
+
Coagulase test
α-type
β. Type
Type of Haemolysis on
bloodagar
Table.3 Biochemical Tests for Identification of E. coli, P. aeruginosa and
C. diversus isolates
Biochemical test
E. coli
P. aeruginosa
C. diversus
Indole
+
_
+
Methyl red
+
_
+
Voges proskauer
-
_
_
Citrate utilization
-
+
+
Urease production
-
+
V
Oxidase
-
+
_
Catalase
+
+
Klegller
Gas production
H2S production
Slope
Bottom
+
-
Acid
Acid
+
+
Alkaline
Alkaline
V
_
Acid
Acid
Motility
+
+
+
Table.4 Ability of Bacterial Isolates to Production of L- Glutaminase
Isolates species and
number. *
Total
isolates
counts
Isolates No.
Glutaminase
production assay
(Zone index)
Staphylococcus aureus
63
7, 14, 42, 56
2.0
15, 27, 51
1.5
Staphylococcus
epidermis
24
5, 12
1.75
1 6, 23
1.5
Pseudomonas
aeruginosa
23
15,23
3.0
1, 9, 20
2.75
Escherichia coli
56
7
0.5
3, 27, 40,52,22,37
0.25
Citrobacter diversus
12
1, 8
1.25
10
1.0
* Bacterial isolates numbers that’s not mention means not produced L-glutaminase

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
223
Table.4-6 L-Glutaminase Activity (IU/Ml) for Bacterial Isolates from Wounds Source
Isolates species and number. *
Isolates No.
Glutaminase activity
(IU/ml)
Staphylococcus aureus
7, 14, 42, 56
12.43 to 18.26
15, 27, 51
8.6 to 10.7
Staphylococcus epidermis
5, 12
11.5, 12.2
1 6, 23
6.9, 8.5
Pseudomonas aeruginosa
15, 23
12.3, 14.0
1, 9, 20
8.5 to 11.7
Escherichia coli
7
18.5
3, 27, 40,52
11.0 to 14.3
22, 37
7.2, 9.4
Citrobacter diversus
1, 8
10.0, 13.8
* Bacterial isolates numbers that’s not mention means not produced L-glutaminase
L-glutaminase have been reported in many
microbial species but their biochemical, and
enzymatic, substrate specificity, molecular
weight and antitumor activities vary with
genetic nature and cultural conditions which
optimized by investigators for various
microorganisms as for filamentous fungi by
(Nathiya et al., 2012).The enzyme activity
for E.coli estimated by (Hughesd and
Williamsodn, 1952), the optimum activity of
the L-glutaminase A and B which produce
by E.coli depends on PH, Glutaminase A
have optimal activity at PH about 5, such
enzyme would be unsuitable for clinical
application where they would be required to
be use at PH above 7 as that used by
(Roberts et al., 1970). Whereas the
glutaminase B have maximum activity at pH
above 7 (Prusiner, 1975).
The maximum yield of L-glutaminase from
Pseudomonas aeruginosa and Serratia
marcescens obtained following optimization
of fermentation process by (Rashmi et al.,
2012). Also L-glutaminase production from
aerobic gram positive filamentous bacteria
Streptomyces griseus under optimized
conditionwas reported by (Suresh Kumar et
al., 2013). With maximum activity of
45IU/ml. However, Tullimilli et.al.
(Tullimilli et al., 2014), reported the
maximum activity of L-glutaminase
produced by fungal strain Mucor racemosus
at 969.23 IU/mlafter optimizing culture
conditions. It have been concluded from
these results that E.coli No.7 has potential
for large scale production of L-glutaminase
enzyme for use in industrial and
pharmaceutical applications.
References
Athira, R.N., Elizebeth, T., Narendra, T.,
Sheik Tanweer Ahmed, Shankar
Kumar Gupta, Manoj Chaudary,
Siddalingeshwara, K.G., Pramod, T.
2014. Investigation on the production
of L-glutaminase from Pseudomonas
stutzeri strain under solid state
fermentation using various agro
residues. J. Drug Delivery &
Therapeutics; 4(2): 81–85.
Elshafei, A.M., Hassan, M.M., Ali, N.H.,
Abouzeid, M.A., Mahmoud, D.A.,
Elghonemy, D.H. 2014. Purification,
Kinetic properties and antitumor
activity of L-glutaminase from
Penicillium brevi compactum NRC
829. British Microbiol. Res. J., 4(1):
97–115.

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
224
Gulati, R., Saxena, R.K., Gupta, R.A. 1997.
Rapid plate assay for screening of L-
asparginase producing
microorganisms. Lett. Appl.
Microbiol., 24: 23–26.
Hartman, S.C. 1968. Glutaminase of
Escherichia coli. Purification and
general catalytic properties. J. Bio.
Chem., 243: 853–863.
Holcenberg, J.S. 1982. Enzyme Therapy:
Problems and Solutions. Ann. Rev.
Biochem., 51: 795–812.
Holt, J.G., Grieg, N.R., Sneath, P.H.A.,
Staley, J.T., Williams, S.T. 1994.
Bergey’s manual of determinative
bacteriology 9th Ed. Williamsons and
Wilkins, Baltimore.
Hughesd, E., Williamsodn, H. 1952. Some
properties of the glutaminase of
Clostridium welchii. Biochem. J., 51:
45–55.
Imada, A., Igarasi, S., Nakahama, K.,
Isono, M. 1973. Asparginase and
glutaminase activities of
microorganisms. J. Gen. Microbiol.,
76: 85–99.
Iyer, P., Singhal, R.S. 2008. Production of
glutaminase (E.C.2.3.1.5) from
Zygosaccharomyces rouxii: statistical
optimization using response surface
methodology. Biores. Technol., 99:
4300–4307.
Nathiya, K., Sooraj, S.N., Angayarkanni, J.,
Palaniswamy, M. 2012. In vitro
cytotoxicity of L-Glutaminase against
MCF-7 cell lines. Asian J. Pharm.
Clin. Res., 5(2): 171–173.
Oshima, M., Yamamoto, T., Soda, K. 1976.
Further characterization of
glutaminase isozymes from
Pseudomonas aeruginosa. Agri. Biol.
Chem., 40: 2251–2256.
Pruisner, S., Oavis, J.N., Stadtman, E.R.
1976. Regulation of glutaminase B in
E.coli. J. Biol. Chem., 251: 3447–
3456.
Prusiner, S. 1975. Regulation of
glutaminase level in Escherichia coli.
J. Bacteriol., 123(3): 992–999.
Rashmi, A.M., Gopinath, S.M., Krishan
kumar, Narasimha Murthy, T.P.
2012. Optimization of submerged
fermentation process for L-
glutaminase produced by
Pseudomonas aeruginosa BGNAS-5.
Int. J. of Latest Res. in Sci. and
Technol., 1(4): 308–310.
Roberts, J., Holcenberg, J.S., Dolowy, W.C.
1970. Antineoplastic activity of
highly purified bacterial glutaminase.
Nature, 227: 1136–1137.
Sabu, A., Sukumaran, R.K., Muthusamy, C.
2002. L-glutaminase as a therapeutic
enzyme of microbial origin, Methods
in Biotechnology. Vol. 17, Microbial
enzymes and Biotransformation,
Human Press Inc.
Sajitha, N., Vasuki, S., Suja, M., Kokilam,
G., Gopinath, M. 2013. Screening of
L-glutaminase from Seaweed
endophytic fungi. Int. Res J Pharm.
App Sci., 3(5): 206–209.
Saptarshi, S.D., Lele, S.S. 2011.
Localization and disruption kinetics
of L-asparginase from Erwinia
carotovora cells by high power
ultrasound. Chem. Biochem. Eng. Q.,
25: 65–73.
Scopes, R.K. 1987. Protein purification. 2nd
ed. Springer. Velag. New York.
Soda, K., Oshima, M., Yamamoto, T. 1972.
Purification and properties of
isozymes of glutaminase from
Pseudomonas aeruginosa.
Biochemistry Biophysics Res.
Communications, 46: 1278–1284.
Suresh Kumar, S., Muthuvelayudham, R.,
Viruthagiri, T. 2013. Medium
optimization for production of L-
Glutaminase (E C.3.5.1.2) by
Streptomyces griseus. IJSEA, 2(1): 1–
5.

Int.J.Curr.Microbiol.App.Sci (2016) 5(4): 218-225
225
Suresh Kumar, S., Muthuvelayudham, R.,
Viruthagiri, T. 2013. Statistical
Optimization based Production of L-
Glutaminase (E C.3.5.1.2) by Serratia
marcescens under submerged
Fermentation. Res. J. Chem. Sci.,
3(6): 43–53.
Tullimilli, A., Sankar, J, Bondili, P.B.,
Ramachandra, R. 2014. Studies on
isolation, screening and molecular
characterization of L-glutaminase
producing marine isolates, IJSID,
4(1): 33–44.
How to cite this article:
Gulbahar F. Karim and Karkaz M. Thalij. 2016. Determination of L-Glutaminase Activity by
Some Bacterial Species. Int.J.Curr.Microbiol.App.Sci. 5(4): 218-225.
http://dx.doi.org/10.20546/ijcmas.2016.504.027