ISOLATION,CHARECTERIZATION AND IDENTIFICATION OF L-GLUTAMINASE

PRODUCING ORGANISM FROM SOIL SAMPLE

SHREYA MODIROLL NO- 11

Guided and Checked By: Dr. S. A. Bhatt Sir

CONTENT

• Introduction • Materials and Method• Result & Discussion • Conclusion• References

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.

MICROBIAL SOURCES OF L-GLUTAMINASEActinetobacter glutaminisificans

T Holchenberg, 1985

Bacillus licheniformis

T Cook et al., 1981

Erwinia cartowora T Wade et al., 1971

Microccus luteus M Moriguchi et al., 1994Pseudomonas 7A T Sabu, 2000a

Vibrio costicola M Nagendraprabhu and

Chandrasekaran, 1996

FUNGI Actinomucor elegans

T Chou et al., 1993

Actinomonas taiwanensis

T Chou et al., 1993;Lu et al., 1996

Aspergillus awamori

T Jones and Lovitt, 1995

Aspergillus oryzae

T Choi et al.,1991;Yano et al., 1991;Sabu, 2000a

Aspergillus sojae T Ushijima et al., 1987

T= Terrestria

YEAST Candida sp T Sabu, 2000aCandida utilis T Kakinuma et al.,

1987

Saccaromyces cerevisiae

T Abdumalikov et al.,1967

Cryptococcuslaurentii

T Kakinuma et al., 1987

Rhodosporidium toruloides

T Ramakrishnan andJoseph, 1996

Torulopsis candida

T Kakinuma et al., 1987

T= Terrestrial

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

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)

• 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.

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

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.

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,.

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.

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.

Conti..

• 4thday workThis 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.

• 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.

Result of isotion of L-Glutaminase producer organism

Colony MorphologySize SmallShape RoundMargin EvenElevation ConvexSurface SmoothPigment Pale yellowConsistency ViscousTransparency Transparent

Result of Gram’s staining Size Small

Shape RodGram reaction NegativeOrganism 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.

BIOCHEMICAL TESTS Sr.No TEST RESULT1 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

2 Methyl red test Negative3 Vogas-proskauer’s test Negative4 Citrate utilization test Positive5 Indol production Negative6 Hydrogen sulphide production test Positive7 Decarboxylation [moeller’s] test Positive8 Urea hydrolysis test Positive9 Nitrate reduction test Positive10 Ammonia production test Positive11 Starch hydrolysis test Negative12 Casein hydrolysis test Negative13 Lipid hydrolysis test Positive14 Catalase test Positive15 Dehydrogenase test Negative16 Litmus milk test Positive

PHYSICOCHEMICAL ANALYSISNo Parameter Method Used Result

1 Colour Munsell’s chart Brown

2 pH pH Meter 7.9

3 Calcium carbonate

Rapid Titration method 67mg

4 Organic carbon

Walkley and Black’s method 2240 mg/lit

5 Phosphorus Fiske and Subbarow’s Method

0.040g%

6 Sulfur Spectrophotometric method 0.503g%

7 Total hardness EDTA titration method 216.996mg

8 Inorganic nitrogen

Micro-Kjeldhal method -

9 Chloride Mohr’s method 31.24g%

Colony on DCA agar Plate Colony on N agar Plate

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.

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.

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.

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