2. CONTENT
• Introduction
• Materials and Method
• Result & Discussion
• Conclusion
• References
3. INTRODUCTION
• L-glutaminase is an amidohydrolase which catalyses the
hydrolytical deamination of L-glutamine resulting in the
production of L-glutamic acid and ammonia. L-Glutaminases
are ubiquitous in the biological world and organisms ranging
from bacteria to human beings have the enzyme. L-
Glutaminase has a central role in mammalian tissues .
• A parallel interest on microbial L-glutaminases stemmed
from its applications in food flavouring, especially in the
soy sauce and related industries of the orient, which
initiated the quest for industrial sources of the enzyme.
With the development of biotechnology, microbial L-
glutaminases found newer applications in clinical
analysis and even in manufacture of metabolites.
4. MICROBIAL SOURCES OF L-GLUTAMINASE
Actinetobacter T Holchenberg, 1985
glutaminisificans
Bacillus T Cook et al., 1981
licheniformis
Erwinia T Wade et al., 1971
cartowora
Microccus luteus M Moriguchi et al., 1994
Pseudomonas 7A T Sabu, 2000a
Vibrio costicola M Nagendraprabhu and
Chandrasekaran, 1996
5. FUNGI T= Terrestria
Actinomucor T Chou et al., 1993
elegans
Actinomonas T Chou et al., 1993;
taiwanensis Lu et al., 1996
Aspergillus T Jones and Lovitt,
awamori 1995
Aspergillus T Choi et al.,1991;
oryzae Yano et al., 1991;
Sabu, 2000a
Aspergillus sojae T Ushijima et al.,
1987
6. YEAST T= Terrestrial
Candida sp T Sabu, 2000a
Candida utilis T Kakinuma et al.,
1987
Saccaromyces T Abdumalikov et al.,
cerevisiae 1967
Cryptococcuslaure T Kakinuma et al.,
ntii 1987
Rhodosporidium T Ramakrishnan and
toruloides Joseph, 1996
Torulopsis T Kakinuma et al.,
candida 1987
7. APPLICATION OF L-GLUTAMINASE
• Food industry
• Used as Biosensors
• Manufacture of fine chemicals
• For the treatment of Cancer, HIV, etc..
• Used as antitumor drugs
• Online monitoring of fermentation process
8. MATERIALS AND METHODS
• Different media for isolation of L-glutaminase
producer.
• There are different media uses for different organism:
• For organism from a marine source, the medium
composition:
• Nutrient Broth (Himedia, India) -13g
• NaCI -10g*
• L-glutamine -lg.
• Distilled water - 1000ml
• pH -6
• Final NaCI concentration in the medium - 1.5% (w/v)
9. • For T. Koningii: isolated using wheat bran of 70%
initial
• moisture content, initial pH 7.0, supplemented
with D-glucose (1.0%)
• and L-glutamine (2.0% w/v)
• Components of MGA (gram/litre) :-include 0.5
Dextrose; 0.5 KCl; 0.5 MgSO4; 1.0 KH2PO4; 0.1
FeSO4; 0.1 ZnSO4; 25 NaCl; 10 Lglutamine; 0. 25
phenol red in which Lglutamine act as carbon and
nitrogen source and phenol red act as pH
indicator.
10. CONTI…
• For actinomycete strains: Components of MSG
medium include (grams/litre) 1.0 KH2Po4; 0.5 MgSo4;
0.1 CaCl2; 0.1 NaNo3; 0.1 tri sodium citrate; 25 NaCl;
10 glucose.
• (SWG) Sea water glutamine medium: L-Glutamine 20g
D-Glucose10g
Aged Sea water 1000, pH 8. medium (Peptone 5g,
Yeast extract 1g, Nacl as per 2.45g, Aged Sea water:
• Pencillium expansum&Aspergillus wentii;-Modified
Czapek Dox’s medium
11. ISOLATION OF L-GLUTAMINASE
PRODUCING MICROORGANISM FROM SOIL
• Specific type of organism require the specific
growth factor which provide by different
enrichment media. Likewise L-glutaminase
producer also require specific enrichment media
• Eg: Minimal glutamine agar,
• Luria broth,
• Modified Czapek dox’s medium .
• So we are using a Minimal glutamine agar media
for isolation of L-glutaminase producer.
12. 1stday work
• Requirements:
• 100 ml of Minimal glutamine broth ,250
mlerlenmeyer flask ,different soil sample.
• Preparation of enrichment culture:
• Take a 100 ml of Minimal glutamine broth in
250 ml erlenmeyer flask and add 10 gm of soil
sample and then it incubate at 37˚c for 24-48
h. and perform same procedure for each
collected soil sample 250 rpm,.
13. 2ndday work
• Requirements:
• Dilution tube, Minimal glutamine agar plate,24-48 h old active
culture(enrichment culture).
• Procedure:
• It involve the double dilution method (10-2,10-4 )
• Preparation of 10-2: [Take 5 ml distilled water and 0.05 ml
enrichment culture.]
• Preparation of 10-4:[Take 5 ml distilled water and 0.05 ml from
10-2.] .
• Then make a two Minimal glutamine agar plate of 10-2 and
another two plate for 10-4.And spread 0.1 ml from each dilution
on their respective Minimal glutamine agar plate for isolation of
organism.Then incubate plate at 37˚C for 24-48 hrs.
14. 3rdday work
• After incubation pick single isolated colony
from this plate and streak on same agar plate
for isolation.And incubate the plate for 24
hrs.and also in nutrient agar plate.
15. Conti..
• 4thday work
This colony characteristics show in table.
• Then this unknsown organism preserve on
nutrient agar and Minimal glutamine agar slant
and plate.
• 5thday work:
• Pick up the isolated colony from this slant and
make culture suspention and perform a gram
staining from it. and then note down cellular
morphology from gram staining.
16. • 6th day work:
• Al l The biochemical test are perform for identification
of organism. Examples of biochemical reactions are
oxidation, fermentation, hydrolysis and degradation.
Products of biochemical reactions cause changes to the
medium that you have inoculated the organism with
E.g. An acidic product ↓pH of a medium .pH indicator
in the medium will exhibit a color change indicating
that an exoenzyme is released by bacteria that cause
the product to be formed.
17. Result of isotion of L-Glutaminase
producer organism
Colony Morphology
Size Small
Shape Round
Margin Even
Elevation Convex
Surface Smooth
Pigment Pale yellow
Consistency Viscous
Transparency Transparent
18. Result of Gram’s staining
Size Small
Shape Rod
Gram reaction Negative
Organism Short rod
From performing gram staining the cell show pink in
color and rod in shape and short & long chain so the
organism are gram negative.
19. BIOCHEMICAL TESTS
Sr.No TEST RESULT
1 Carbohydrate fermentation test
- Glucose Acid produce
- Sucrose Acid produce
- Maltose Acid produce
- Mannitol Acid produce
- Xylose Acid produce
- Arabinose Acid produce
Test H2S Gas Slant Butt
TSI test Positive Negative Alkaline Acidic
20. 2 Methyl red test Negative
3 Vogas-proskauer’s test Negative
4 Citrate utilization test Positive
5 Indol production Negative
6 Hydrogen sulphide production test Positive
7 Decarboxylation [moeller’s] test Positive
8 Urea hydrolysis test Positive
9 Nitrate reduction test Positive
10 Ammonia production test Positive
11 Starch hydrolysis test Negative
12 Casein hydrolysis test Negative
13 Lipid hydrolysis test Positive
14 Catalase test Positive
15 Dehydrogenase test Negative
16 Litmus milk test Positive
21. PHYSICOCHEMICAL ANALYSIS
No Parameter Method Used Result
1 Colour Munsell’s chart Brown
2 pH pH Meter 7.9
3 Calcium Rapid Titration method 67mg
carbonate
4 Organic Walkley and Black’s method 2240 mg/lit
carbon
5 Phosphorus Fiske and Subbarow’s 0.040g%
Method
6 Sulfur Spectrophotometric method 0.503g%
7 Total hardness EDTA titration method 216.996mg
8 Inorganic Micro-Kjeldhal method -
nitrogen
9 Chloride Mohr’s method 31.24g%
23. CONCLUSION
• L- glutaminase producing organism associated with fertile
soil were evaluated, characterized, and identified. The
organism which produce l-glutaminase enzyme is a
Member of Enterobacteriaceae family identified and
characterized by performing gram’s staining and different
biochemical test.
• The organism appear as small, round, pale yellow , smooth
and gram negative short rod .
• These identities of isolate were based on morphological,
cultural, physiological and biochemical characteristics of
Enterobacteriaceae family presented in bergey’s manual
of systematic bacteriology.
24. CONTI…
• By performing all the biochemical test we concluded
that the unknown organism is a members of
Enterobacteriaceae family beacause VP, MR,Indol test
are negative. And also gelatin liquification, Urea
hydrolysis , catalase ,ammonia production, nitrate
reduction , phenylalanine deamination are positive.
• The different minerals also present in soil sample are
carbon , nitrogen, inorganic phosphate, sulphate. And
also we estimate the gm% of this mineral present in soil
sample.
• So it can be concluded that the isolate organism is a
member of Enterobacteriaceae family.
25. REFERENCES
• Kashyap P, Sabu A, Pandey A, Szakacs G, Soccol CR (2002).
Extracellular L-glutaminase production by Zygosaccharomyces
rouxii under solid-state fermentation. Proc. Biochem., :307-
312
• Klein M, Kaltwasser H, Jahns T (2002). Isolation of a novel,
phosphate activated glutaminase from Bacillus pasteurii.
FEMS Microbiol. Lett., 206: 63–67.
• Prabhu GN, Chandrasekaran M (1997). Impact of process
parameters on L-glutaminase production by marine Vibrio
costicola under solid state fermentation using polystyrene as
inert support. Proce Biochemistry.: 285-289.
