Optimization of process parameters for the production of
glutaminase enzyme from Erwinia at flask level.
*Pushpinder Paul.
Department of Biotechnology,
M.G.C. Fatehgarh Sahib,Punjab
Mb-9762046125
Email Id-pushpinder_pushp@yahoo.co.in
Abstract:
This purpose of study was to determine parameter
optimization for the production of Glutaminase enzyme from Erwinia at flask
level. This study was carried out at
Department of Biotechnology, Mata Gujri College, Fatehgarh Sahib, Punjabi
University Patiala For maximum production of glutaminase from free cells of Erwinia, various physical and chemical
parameters were optimized. The microorganism Erwinia which is a source of glutaminase was procured from
Institute of Microbial Technology, Chandigarh and was grown on specified medium.
The media was sterilized by autoclaving for 15 minutes at 15 psi/cm2.
The microorganism was maintained on agar slants and kept in refrigerator at 4.0
+ 0.5 0C for further
use and was sub-cultured after every 20 days. The viability check of microorganism
was done by gram staining and tryphan blue. The work utilized Beef extract,
Peptone, Yeast extract, Sodium chloride and Water as composition of growth
medium for the production of glutaminase enzyme from Erwinia. The parameters
such as Temp., Ph, Time and Inducer concentration were optimized.The enzyme
activity was determined by Nessler’s reagent method and protein concentration
was determined by Bradford method. It was concluded that Maximum production of
glutaminase enzyme from Erwinia was observed at temp.-25◦C, pH-6.5 & Time
after-48 hours. At these optimized parameters enzyme activity and Protein
concentration was found maximum.
Key Words:
Glutaminase Enzyme, Erwinia, Parameters optimization
Introduction:
The protein glutaminase is traditionally
considered as a mitochondrial enzyme, playing a key role in the energy and
nitrogen metabolism of mammalian cells. The microbial enzymes are found to play a major
role in the diagnosis, curing, biochemical investigation, and monitoring many dreadful diseases.
Microorganisms represent an excellent source of many therapeutic enzymes owing
to their broad biochemical diversity and their susceptibility to genetic
manipulation.
Glutamine, out of the 20 amino acids is commonly found in animal
proteins which supplies energy and substrate to the host after severe stress.
Only the l -stereoisomer is found to occur in mammalian protein. The liver shows net glutamine uptake
after a protein-containing meal during uncontrolled diabetes, sepsis and
short-term starvation but changes to net release during long-term starvation
and metabolic acidosis. Glutamine structure is identical to that of glutamic acid
except that the acidic side-chain carboxyl group of glutamine has been
coupled with ammonia, yielding an amide. The glutamic acid-glutamine
interconversion is of central importance to the regulation of the level of toxic ammonia in the body. Thus, it is not
surprising that when the concentration of the amino acids of blood plasma are
measured, glutamine is found to have the highest value. Glutaminase can be
isolated from different microbial, plant and animal sources. The microbial sources
from which glutaminase has been extracted are Bacillus licheniformis A5,Micrococcus luteus K-3, Erwinia species.
Erwinia is a genus of Enterobacteriaceae containing mostly plant pathogenic
species which was named by the first phytobacteriologist, Erwin Smith. It is a
gram negative bacterium related to E.coli, Shigella,
Salmonella
and Yersinia.
Hence this work was carried out to
determine the parameter optimization for the production of Glutaminase enzyme
from Erwinia.The study was based on optimization of parameters such as Time,
Temp., pH etc. for the production of Glutaminase.
Materials & Methods:
1.
Determination of enzyme activity
Glutaminase activity can be defined as the mM of glutamine formed per ml
of enzyme solution in one
minute. Glutaminase activity was determined spectrophotometrically by Nessler’s reagent.
Reagents:
1)Prepared stock solution of L-glutamine (100mM) by
dissolving 1.465g of L-glutamine in 100ml distilled water.
2)TCA (tri-carboxylic acid) – 2.45g in 100ml distilled
water
3)Phosphate buffer – It was prepared by setting pH of K2HPO4
(1.74g in 100ml distilled water) to 7 by KH2PO4 which
is prepared by dissolving 1.36g in 100ml distilled water.
4) Nesseler’s reagent(S D fine-Chem. Ltd.).
Procedure:
The
different concentrations ranging 200, 400, 600, 800, 1000, 1200, 1400, 1600µl
of L-glutamine (100 mM) were taken in 8 test tubes and final volume was made to
4000µl by adding distilled water. The blank was maintained by using distilled
water.3ml of Phosphate buffer having pH 7 was added to each test tube including
blank and were kept at 37 0C in an incubator for 15 minutes.5ml of
Nessler’s reagent was added. To the above solution, 0 .4ml of TCA was
added.O.D. was taken at 630 nm.The concentration of ammonium ions formed was
observed from the standard curve of
ammonium ions and enzyme activity was calculated.
2.Determination
of protein concentration
It was done by the procedure
given by Bradford, 1976. The protein in a sample was calculated by using this method .This
method was found to be quite sensitive and was able to detect concentrations as
low as 20 mg of proteins.
Reagents:
BSA (Bovine
serum albumin) – 0.1g in 100ml distilled water. (Genei).
Bradford’s
reagent. (Genei).
Procedure:
The different volumes of BSA were taken in six different
test tubes.The final volume was made 200µl by putting distilled water. Then,
3ml of Bradford’s reagent was added in each test tube.
The test tubes were incubated at room temperature for 10
minutes.Took the absorbance at 480nm.
3. Optimization of time (growth curve) for glutaminase production from Erwinia:
To check the optimum time of maximum enzyme production from free cells , cells were inoculated(10%) in the production medium and samples were collected after 6 hours of time interval and in each sample enzyme activity was observed.
4.
Optimization of pH of the production medium for glutaminase production from
Erwinia
The
optimum pH for glutaminase production from free cells of Erwinia was determined by dividing the batch in different pH
groups. The various pH ranges selected were 2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8
respectively.
5.
Effect of temperature on glutaminase production:
The effect of temperature on glutaminase production was
determined by incubating the flasks at different temperatures. The temperature
ranges selected were 20, 25, 30, 35, 40, 45, and
50°C.
Results & Discussion
1 Optimization of process parameters for
production of glutaminase from Erwinia
1.
I Standard curve for Glutamine
The standard curve for Glutamine was prepared using
Nessler’s method. The absorbance of different concentrations of Glutamine given
in Table 1.1were recorded. It was found that conc. of glutamine is directly
proportional to the absorbance noted.
Table:
1.1 Absorbance for different concentration of glutamine
Fig.1.1 Standard curve of
Glutamine
1.2
Standard curve for protein (BSA)
The protein concentration in a
sample was calculated using the Brad ford method. This method is quite
sensitive and can detect quantity as low as 20 mg of proteins. The absorbance for
different concentration of protein(BSA) were noted. It was found that BSA
conc.was directly proportional to the absorbance noted.
Table:
1.2 Absorbance for different concentration of protein (BSA).
Fig.
1.2 Standard curve of protein
1.3
Optimization of time for glutaminase production from Erwinia species:
The
microorganism produce
different enzymes at different times of its life cycle. To check optimum time for maximum
production of
glutaminase from free cells of Erwinia, samples were collected after
different time intervals and enzyme activity was observed. It was observed that
enzyme production increased with
time
(0.003-.04 U/ml) and there was maximum production after 48 hours. After this period there was almost constant rate of enzyme production
was observed up to 60 hours. In Z.rouxi
the maximum production was also obtained after 48 hours.(Kashyap,et al.,2002)
Table1.3 For optimization of time
for glutaminase production from Erwinia.
Fig.1.3
Standard curve for optimization of time for glutaminase production
1.4
Optimization of pH for glutaminase production:
To observe the effect of pH on glutaminase
production
the
fermentation was carried at various pH ranging from 2,2.5,3
,3.5,4,4.5.5,5.5,6,6.5,7,7.5,8. It was observed that optimum enzyme production
was at pH 6.5 as compared to glutaminase of Actinomucor taiwanensis which was found to be stable between pH
6.0-8.0. (Kirzimura et al.,1969).
Various researchers found the maximum enzyme activity lies between the
range 5-9. The enzyme activity of Beauweria (Sabu et al.,2ooo) was observed to
be quite higher i.e. at pH 9 whereas enzyme activity was also found at lower
pH.
Table
1.4 Optimization of pH for glutaminase
production from Erwinia.
Fig.
1.4 Standard curve for optimization of pH for glutaminase production
1.5
Effect of temperature on glutaminase production:
The effect of temperature on
glutaminase production was determined by incubating the culture broth at
different temperature ranging from 20 to 50ْC.
Table:
1.5 Effect of temperature on glutaminase production
CONCLUSION:
From this study it was cleared that Maximum production of
glutaminase enzyme from Erwinia was observed at temp.-25◦C, pH-6.5 & Time
after-48 hours. These results obtained serves as an established database for
the Glutaminase production from Erwinia. As Glutaminase has many applications
in industries and Glutaminase or glutaminase-asparaginase enzymes have
received scant attention from investigators compared to other established
industrial enzymes because the enzyme needs inorganic phosphate in invitro
activity and plays a key role in several physiological processes, such as renal
ammonia genesis, hepatic urea genesis and synthesis of neurotransmitter
glutamate in brain which is found to be helpful in curing many neurodegenerative diseases .This study must
be very helpful for such investigators.
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