Production and optimization of L-Glutaminase enzyme from hypocrea jecorina pure culture

Article abstract of DOI:10.1080/10826068.2012.741641

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver-Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.

Full text of DOI:10.1080/10826068.2012.741641

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PRODUCTION AND OPTIMIZATION OF L-
GLUTAMINASE ENZYME FROM Hypocrea
jecorina PURE CULTURE
a
a
Dilara Bülbül & Emine Karakuş
a
Department of Chemistry , Faculty of Science and Arts, Yildiz
Technical University , İstanbul , Turkey
Accepted author version posted online: 12 Dec 2012.Published
online: 06 Mar 2013.
To cite this article: Dilara Bülbül & Emine Karakuş (2013) PRODUCTION AND OPTIMIZATION OF
L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE, Preparative Biochemistry and
Biotechnology, 43:4, 385-397, DOI: 10.1080/10826068.2012.741641
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Preparative Biochemistry & Biotechnology, 43:385–397, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6068 print/1532-2297 online
DOI: 10.1080/10826068.2012.741641
PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME
FROM Hypocrea jecorina PURE CULTURE
Dilara B u¨ lb u¨ l and Emine Karaku s¸
Department of Chemistry, Faculty of Science and Arts, Yildiz Technical University,
˙
Istanbul, Turkey
&
L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that
catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently,
L-glutaminase has received much attention with respect to its therapeutic and industrial applica-
tions. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production
of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and micro-
organisms, including bacteria, yeasts, and fungi. This study presents microbial production of glu-
taminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions
and calculation of kinetic parameters of the produced enzyme. The optimum values were determined
by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value
ꢀ
was determined as 8.0 and optimum temperature as 50 C for the glutaminase enzyme. The Km and
Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture
were determined as 0.491 mM for Km and 13.86 U=L for Vmax by plotted Lineweaver–Burk graph-
ing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal
and storage stability.
Keywords enzyme production, glutaminase, Hypocrea jecorina, L-glutamine, Nessler
reagent
INTRODUCTION
The reaction of glutaminase plays a major role in the nitrogen
metabolism of both prokaryotes and eukaryotes. L-Glutaminase
(
L-glutamine amidohydrolase, E.C 3.5.1.2) is a hydrolytic enzyme that
hydrolyzes L-glutamine to L-glutamic acid and ammonium ions. It has an
important role in the nitrogen metabolism. Especially because of its phar-
maceutical and food industrial applications, L-glutaminase has attracted
Address correspondence to Emine Karaku s¸ , Yildiz Technical University, Faculty of Science and
Arts, Department of Chemistry, Esenler, I˙ stanbul, Turkey. E-mail: karakus@yildiz.edu.tr

386
D. B u¨ lb u¨ l and E. Karaku s¸
much attention in recent years. In the food industry, L-glutaminase is used
as a flavor-enhancing agent that acts by increasing glutamic acid content in
food due to its catalyzed hydrolysis reaction of L-glutamine to L-glutamic
[
1,2]
acid and ammonium.
It also used in enzyme therapy for cancer, espe-
[3,4]
cially for acute lymphocytic leukemia.
production of L-glutaminase, its production is very important. Another
important usage of L-glutaminase is its application in biosensors for
Due to demand for large-scale
[5]
[6–11]
monitoring the glutamine levels in mammalian and hybridoma cells.
L- Glutamate generally has an interference effect on L-glutaminase biosen-
sors. The biosensors prepared by using L-glutaminase enzyme allow moni-
toring glutamine levels in mammalian and hybridoma cell cultures without
[12]
the need for separate measurement of glutamic acid.
L-Glutaminase is widely distributed in animal tissues, plants, and
[13]
microorganisms, including bacteria, fungi, and yeasts.
L-Glutaminase
production by various bacterial genera under submerged culture con-
[
14–18]
ditions was extensively studied.
Furthermore, submerged cultures
[
15]
[19]
[16]
of Verticillium malthousei,
Aspergillus sojae,
and Penicillium notatum
A. Nidulans,
Tilachlidium
[17]
[18]
humicola,
production.
have been used for L-glutaminase
Although Hypocrea jecorina (anamorph Trichoderma reesei) is an indust-
rially important producer of cellulase for an applications portfolio that
includes pulp and paper processing, food and feed processing, textile
manufacturing and modification, and detergents=cleaners formulation, it
[20]
is potentially used in all areas of enzyme production.
Our study presents microbial production and optmization of gluta-
minase enzyme from Hypocrea jecorina. The optimum parameters of the
produced enzyme, such as optimum pH, optimum temperature, buffer
concentration, thermal stability, and storage stability of enzyme, are also
determined.
MATERIALS AND METHODS
Materials
The Hypocrea jecorina microorganism used in this study was obtained
from the Institute of Biochemical Technology and Microbiology, Vienna
Technical University. L-Glutamine, glucose, potato dextrose agar (PDA),
bovine serum albumin (BSA), Coomassie brilliant blue G-250, ethanol,
o-phosphoric acid, trichloroacetic acid (TCA), KH PO , MgSO ꢁ 7H O,
2
4
4
2
KCl, NaCl, NaOH, NaH PO Na HPO , KI, and HgCl were purchased
2
4,
2
4
2
from Sigma Chemical (St. Louis, MO). All chemicals used in this study were
of analytical grade and were used without further purification. The absor-
bance measurements were carried out with the Agilent ultraviolet–visible

Production of L-Glutaminase Enzyme
387
(
UV-Vis) spectrophotometer. The Sartorius PB11 model pH meter was used
for pH measurements. The incubation, sterilization, heating, cooling and
storing procedures used the VWR-incubating mini shaker, Clifton brand
water bath, Certoclav brand autoclave, Memmert brand oven, and Ar c¸ elik
brand refrigerator, respectively. The GFL brand water destillation device
was used to obtain distilled water.
Fermentation Medium and Culture Conditions for
Microorganisms
To sustain growth and vitality of the Hypocrea jecorina microorganism,
potato dextrose agar (PDA) was prepared. For this purpose, 39 g potato
dextrose agar (PDA) was dissolved in water and the final solution volume
was completed to 1 L with distilled water. This PDA solution was sterilized
ꢀ
ꢀ
by autoclaving for 20 min at 121 C. After sterilization, it was kept at 45 C
in a water bath and the solution was apportioned into petri dishes and
allowed to cool. The solidified media was stored in the refrigerator
at þ4 C until used.
ꢀ
For preparing PDA slant agar medium, 15 mL of the solution prepared
was apportioned into each tube and, with the lids of the tubes closed, ster-
ilized by autoclaving for 20 min at 121 C. After the sterilization process, the
ꢀ
tubes were allowed to cool, positioned flat. The solidified media was stored
ꢀ
at þ4 C until used.
Hypocrea jecorina was planted into each petri dish containing solidified
culture media with a sterile loop and incubated for 7 days in an oven set
ꢀ
at 30 C. After an incubation period of 7 days the petri dishes containing
ꢀ
produced H. jecorina were covered with parafilm and stored at 4 C until
used. This procedure was repeated when fresh H. jecorina was needed. To
ꢀ
maintain vitality of the mold strains stored at 4 C, they were transferred
to fresh media once a month.
To produce crude L-glutaminase enzyme extract from Hypocria jecorina,
we used our modified fermenting liquid media method proposed from the
[
2]
El-Sayed method. The medium contains these components (in grams per
liter): glucose as carbon source 1.0, KH PO 0.1, MgSO ꢁ 7H O 0.05, KCl
2
4
4
2
0.05, NaCl 5.0, and L-glutamine as nitrogen source 4.0. After sterilization
of 100 mL of the solution containing these liquid media components at
ꢀ
ꢀ
1
21 C for 20 min, the solution was incubated in a water bath at 30 C for
3
0 min; 2 mL of Hypocria jecorina pure culture was added to this sterilized
ꢀ
solution medium and incubated at 30 C and 150 rpm in a shaking incu-
bator for 30 min. After incubation for 30 min, we measured L-glutaminase
activity. Experiments and enzymatic assay were performed in triplicate for
each sample.

388
D. B u¨ lb u¨ l and E. Karaku s¸
Optimization of the Culture Condition for L-Glutaminase
Production
To maximize the yield of L-glutaminase from Hypocrea jecorina, several
parameters such as effect of substrate and salt concentrations were investi-
gated. To determine optimum substrate concentration for maximum
L-glutaminase activity, culture medium containing L-glutamine concentra-
tion ranging from 1% to 5% w=v was prepared separately. The effects of
different doses of NaCl (0–15% w=v) to the production medium to maxi-
mize enzyme activity were also studied. The effects of substrate and salt
concentration on the enzyme production were evaluated by measuring
L-glutaminase enzyme activity. The optimized parameter was incorporated
at its optimized level in the all experiments. All the experiments were con-
ducted in triplicate. After incubation of each fermentation sample, the
crude enzyme was prepared.
Quantitative Determination of Glutaminase Enzyme Activity
In this study, we used a method based on measurement of formed
ammonium ions after the enzymatic reaction of L-glutaminase enzyme produ-
[15]
ced from Hypocrea jecorina pure culture.
L-Glutaminase enzyme catalyzed
L-glutamine (L-Gln) to L-glutamic acid (L-Glu) and ammonium ions, and
the latter can react with Nessler’s reagent to produce an orange product:
LꢂGlutaminase
þ
L ꢂ Gln þ 2H O
ꢂ!
L ꢂ Glu þ NH OH
2
4
For this purpose, 0.5 mL of 0.1 M phosphate buffer (pH 8.0), 0.5 mL of
distilled water, and 0.5 mL of produced L-glutaminase enzyme homogenate
were added into 0.5 mL of 0.04 M L-glutamine solution and incubated at
ꢀ
3
7 C for 30 min for enzymatic reaction to occur. After incubation, 0.5 mL
of 1.5 M trichloroacetic acid (TCA) was added to stop the reaction. Then
.1 mL of final solution was added into 3.7 mL of distilled water and Nessler
0
reagent, respectively. After the solution was incubated for 10 min for
interacting with Nessler reagent and ammonium ions formed after
L-glutaminase enzymatic reaction, the solution’s absorbance was measured
at 450 nm.
To determine the activity value of L-glutaminase enzyme, the amount of
þ
the NH ions formed as a result of enzymatic reaction must be determined
4
by drawing ammonium standard graph. The ammonium ions were deter-
mined from a standard curve obtained with ammonium chloride as stan-
dard. For this purpose, 0.2, 0.4, 0.6, 0.8 and 1.0 mg=mL of standard
ammonium chloride solutions were prepared. After adding Nessler reagent
into each tube, the tubes were incubated 10 min at room temperature. The

Production of L-Glutaminase Enzyme
389
absorbance of each tube was separately measured against the blank contain-
ing 3.8 mL distilled water and 0.2 mL of Nessler reagent at 450 nm. The
ammonium standard curve was obtained by plotting absorbance values
against ammonium chloride concentrations.
One unit of glutaminase activity was defined as the amount of enzyme
that liberated 1 mmol of ammonium ions produced per minute at room
temperature. Specific activity is expressed as units per miligram of protein.
Protein Determination
The concentration of the total protein was measured by the Bradford
[21]
method using crystalline bovine serum albumin as the standard.
The pro-
tein concentration was expressed in milligrams per milliliter of crude enzyme.
Effect of pH and Temperature on Produced L-Glutaminase
Activity
The optimum pH value of our produced L-glutaminase enzyme from
H. jecorina was determined for the pH range between 5.0 and 9.0 by using
0.1 M phosphate buffer. The optimum pH value determined was used in all
the other experiments.
L-Glutaminase activity, as a function of temperature under the men-
tioned assay conditions, using different temperatures ranging from 30 to
ꢀ
7
0 C, was controlled by a circulating water bath. The relative activity of
L-glutaminase at a specific temperature was determined spectrophoto-
metrically by addition of the produced enzyme extract to the mixture.
The optimum temperature value obtained from these assays was used in
all the other experiments.
Thermal Stability of L-Glutaminase by H. jecorina
The thermal stability of L-glutaminase was investigated at four different
ꢀ
temperatures between 40 and 70 C for varying periods of time in a
temperature-controlled water bath. Enzyme solution was placed in a pre-
warmed incubator at the specified temperature and 0.5 mL of the sample
portions was withdrawn at various time intervals during 45 min and residual
activity assayed. The stability of the enzyme was expressed as remaining
activity.
Kinetic Constants of L-Glutaminase
The our produced L-glutaminase kinetic parameters from H. jecorina,
the Km and Vmax values, were determined by measuring activities in the

390
D. B u¨ lb u¨ l and E. Karaku s¸
presence of various substrate concentrations between 10 and 100 mM at pH
8
.0. The Km and Vmax values of the enzyme were calculated by using the
[22]
Lineweaver–Burk double-reciprocal plot,
in which the reciprocals of the
initial velocities of L-glutaminase activity were plotted against the recipro-
cals of the concentration of L-glutamine used.
RESULTS AND DISCUSSION
Production of L-Glutaminase from H. jecorina
L-Glutaminase ezyme is widely found in microorganisms such as bac-
teria and fungi. L-Glutaminase production by various bacterial genera
[14,15,23]
under submerged culture conditions was extensively studied.
Glutaminase enzyme plays an important role in nitrogen metabolism.
In recent years, interest in this enzyme has increased considerably due to
usage in therapeutic and industrial areas. Besides being used as antileuke-
mia agent, it plays the role of taste enhancer in food. In common,
L-glutaminase activity has been observed in plants, animal tissues, and
[24]
microorganisms, including bacteria, yeasts, and molds.
Our study aimed to produce L-glutaminase enzyme from Hypocrea jecor-
ina pure culture, determine optimum conditions, and calculate the kinetic
parameters of produced enzyme. L-Glutaminase is an important enzyme
catalyzing the reaction converting L-glutamine to glutamic acid and
ammonium ions. To determine glutaminase enzyme activity, we measured
ammonium ions released as a result of glutaminase enzyme reaction.
Table 1 summarizes the results of some quantitative parameters of
L-glutaminase produced from H. jecorina.
Optimization of Culture Conditions for L-Glutaminase
Production
In order to determine the effect of substrate (L-glutamine) concentra-
tion on L-glutaminase enzyme production from Hypocrea jecorina pure
culture, the enzyme production was carried out by adding L-glutamine at
different concentrations ranging from 1% to 5%. Activity determinations
TABLE 1 Production of L-Glutaminase from H. jecorina
þ
NH Amount Liberated from
L-Glutaminase Activity Protein Amount
Specific Activity
4
L-Glutaminase Reaction (mg=mL) ꢃ s
(U=mL) ꢃ s
(mg=mL) ꢃ s
(U=mg protein) ꢃ s
4.42 ꢃ 0.5
13.75 ꢃ 0.9
0.341 ꢃ 0.8
40.32 ꢃ 0.5
Note. s, Standard deviation. The data were calculated from the results obtained from triplicate experi-
ments as described.

Production of L-Glutaminase Enzyme
391
were carried out on samples taken at the end of the incubation period. The
obtained results were charted as L-glutamine amount (%) against the total
units (U=mL) (Figure 1).
As shown in Figure 1, when the amount of L-glutamine added is
increased, L-glutaminase enzyme activity also increases. We didn’t add
any other nitrogen source because the microorganism uses L-glutamine
as a nitrogen source and only ammonium ions produced as a result of
L-glutaminase enzymatic reaction are involved in the medium. This situta-
tion is important to correctly determine the enzyme activity. After ingestion
of glucose added as the carbon source in the environment, the micro-
organism was directed to glutamine and used it as both carbon and nitro-
gen source and the production of glutaminase enzyme was carried out. We
found 4% of L-glutamine concentration for optimum L-glutaminase pro-
duction from H. jecorina.
To determine the effect of salt amount added to the culture medium on
L-glutaminase enzyme production from Hypocrea jecorina, the enzyme pro-
duction was carried out by adding salt in different concentratins ranging
from 0% to 15%. At the end of the incubation period, activity determina-
tions were carried out by taking from samples. The total enzyme activity
(
(
U=mL) values were determined and plotted against the salt concentration
%) (Figure 2).
As shown in Figure 2, the produced enzyme activity is highest after
adding 5% salt to the culture media. If more than 5% of salt is added to
the media, the enzyme activity decreases. Weingand-Ziad and collaborators
investigated the effect of salt concentration ranging from 0% to 20% on
FIGURE 1 The effect of L-glutamine concentration on L-glutaminase production from Hypocrea
jecorina. The culture medium contains (g=L) as carbon source glucose 1.0, KH
O 0.05, KCl 0.05, NaCl 5.0, and L-glutamine as nitrogen source 4.0. The data were calculated from the
results obtained from triplicate experiments as described.
2
PO
4
0.1, MgSO
4
ꢁ 7H
2
O

392
D. B u¨ lb u¨ l and E. Karaku s¸
FIGURE 2 The effect of salt (NaCl) concentration on L-glutaminase production from Hypocrea jecorina.
The culture medium contains as carbon source (g=L) glucose 1.0, KH PO 0.1, MgSO O 0.05, KCl
ꢁ 7H
.05, NaCl 5.0, and L-glutamine as nitrogen source 4.0. The data were calculated from the results
obtained from triplicate experiments as described.
2
4
4
2
0
their produced glutaminase enzyme from Lactobacillus rhamnosus micro-
organisms and identified optimum salt concentration of 2.5% for the
[
25]
enzyme production.
In the literature, it was pointed out that glutami-
nase activity is increased in the presence of NaCl concentrations lower than
[25,26]
10%.
Effect of pH and Temperature
The optimum pH and temperature values for produced L-glutaminase
from H. jecorina were determined and the results are presented in Figure 3.
As seen in Figure 3, the activity of L-glutaminase enzyme increased pro-
gressively until the pH value of 8.0. After pH 8.0, the activity decreased very
rapidly. Thus, the optimum working pH of the enzyme obtained from
Hypocrea jecorina was chosen as 8.0. It was reported that the pH optima
for L-glutaminase from Vibrio sp., Debaryomyces spp., and Aspergillus sp.
[26–28]
microorganisms were in the range of pH 7.0–8.5.
The temperature effects on the activities of L-glutaminase enzyme pro-
duced from H. Jecorina were tested at pH 8.0, and L-glutaminase activity as a
function of temperature under our standard assay conditions was deter-
ꢀ
mined using temperatures from 20 to 70 C for 30 min. The results obtained
are shown in Figure 4.
It was observed that L-glutaminase activity increased up to the opti-
ꢀ
mum temperature. The maximum catalytic activity was obtained at 50 C.
L-Glutaminase enzyme activity produced from Lactobacillus rhamnosus was

Production of L-Glutaminase Enzyme
393
FIGURE 3 Effect of pH on L-glutaminase enzyme produced from Hypocrea jecorina. The culture
medium contains as carbon source (g=L) glucose 1.0, KH PO 0.1, MgSO O 0.05, KCl 0.05, NaCl
ꢁ 7H
.0, and L-glutamine as nitrogen source 4.0. The data were calculated from the results obtained from
triplicate experiments as described.
2
4
4
2
5
ꢀ
[25]
also highest at 50 C.
That absence of any loss of enzyme activity at high
temperatures is very important in terms of its analytical and industrial uses.
Thermal Stability of L-Glutaminase from H. jecorina
To determine thermal stability of L-glutaminase from H. jecorina, the
enzyme solution was incubated for various time intervals (15–45 min) at
FIGURE 4 Effect of temperature on L-glutaminase enzyme produced from Hypocrea jecorina. The cul-
ture medium contains as carbon source (g=L) glucose 1.0, KH
.05, NaCl 5.0, and L-glutamine as nitrogen source 4.0. The data were calculated from the results
obtained from triplicate experiments as described.
2
PO
4
0.1, MgSO
4
ꢁ 7H
2
O 0.05, KCl
0

394
D. B u¨ lb u¨ l and E. Karaku s¸
ꢀ
the specified temperature (25–70 C) and the activity was measured at room
temperature. Temperature–stability profiles for L-glutaminase are pre-
sented as relative activity and are shown in Figure 5.
Thermal stability experiments were carried out with L-glutaminase
enzyme from H. jecorina. The enzyme was incubated in the absence of sub-
strate at various temperatures. As shown in Figure 5, the enzyme retained
ꢀ
ꢀ
8
5% of its activity during a 45-min incubation period at 40 C and 50 C.
ꢀ
L-Glutaminase enzyme lost about 5% of its original activity at 70 C for
4
5 min. The activity loss of the enzyme is acceptable. It was shown that there
ꢀ
ꢀ
was not any important activity loss at 60 C and 70 C after 45 min. It can be
said that the enzyme has relatively high thermal stability. This is an impor-
tant feature for enzymes with respect to their bioanalytical and biosensor
applications. It was reported that L-glutaminase produced from Lactobacil-
lus rhamnosus was completely inhibited at 75 C after 15 min of
incubation.
ꢀ
[
25]
ꢀ
We studied all experiments at room temperature (25 C)
without any activity loss of the enzyme. This is a very important factor for
biosensor constructions.
Enzyme Kinetic Studies
The Km and Vmax values for the enzyme at optimal pH and tempera-
[22]
ture were determined by the Lineweaver–Burk plot.
The double-
reciprocal plot was linear (correlation coefficient of 0.7939) over the
FIGURE 5 Thermal stability of L-glutaminase from H. jecorina. The culture medium contains as carbon
source (g=L) glucose 1.0, KH
gen source 4.0. The data were calculated from the results obtained from triplicate experiments as
described.
2
PO
4
0.1, MgSO
4
ꢁ 7H
2
O 0.05, KCl 0.05, NaCl 5.0, and L-glutamine as nitro-

Production of L-Glutaminase Enzyme
395
FIGURE 6 Lineweaver–Burk plots of L-glutaminase from H. jecorina. The culture medium contains as
carbon source (g=L) glucose 1.0, KH PO 0.1, MgSO O 0.05, KCl 0.05, NaCl 5.0, and L-glutamine
2
4
4
ꢁ 7H
2
as nitrogen source 4.0. The data were calculated from the results obtained from triplicate experiments
as described.
L-glutamine concentration range (10–100 mM). The Km and Vmax values
were determined as 0.491 mM and 13.86 U=L for L-glutaminase from
H. jecorina, respectively (Figure 6).
FIGURE 7 Storage stability of L-glutaminase from H. jecorina. The culture medium contains as carbon
source (g=L) glucose 1.0, KH
gen source 4.0. The data were calculated from the results obtained from triplicate experiments as
described.
2
PO
4
0.1, MgSO
4
ꢁ 7H
2
O 0.05, KCl 0.05, NaCl 5.0, and L-glutamine as nitro-

396
D. B u¨ lb u¨ l and E. Karaku s¸
The Km value of L-glutaminase enzyme from Lactobacillus rhamnosus
microorganism was 4.8 mM and the Vmax value of the same enzyme was
[
25]
found to be 13.86 U=L.
The Km value obtained in this study is lower
than in the other studies in the literature. The low Km value for
L-glutaminase enzyme from H. jecorina can be an indicator that the
enzyme’s affinity against L-glutamine is very high.
Storage Stability of L-Glutaminase
In order to determine the storage stability of L-glutaminase, the enzyme
extracts produced from H. jecorina were kept in small Erlenmayer flasks at
ꢀ
4
C for 30 days. The total activity measurements were carried out taking sam-
ples in order to determine the stability during 30 days. Results are shown in
Figure 7. As shown in Figure 6, no decrease in L-glutaminase activity was
observed after 30 days. This indicates that L-glutaminase is very stable.
CONCLUSION
This study presents microbial of glutaminase enzyme production from
Hypocria jecorina pure culture. It is also determined optimum conditions
and calculated kinetic culture parameters of our produced enzyme.
L-Glutaminase enzymes produced from microbial sources are important
with respect to their antileukemic action and as flavor-enhancing agents.
Because of this therapeutic and technological importance, L-glutaminase
enzyme production has biotechnological importance.
ACKNOWLEDGMENT
We gratefully acknowledge the financial support of Yildiz Technical
University Science Research Projects Foundation (project number: 2011-
01-02-KAP07) for this work.
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