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Actinomycetes are known for L-asparaginase activity. In the current study,. 80 actinomycetes were isolated from various soil habitats by serial dilution technique.
Indian Journal of Experimental Biology Vol. 53, December 2015, pp. 786-793

Isolation and identification of actinomycetes for production of novel extracellular glutaminase free L-asparaginase Akansha Saxena*, Ramraj Upadhyay & Naveen Kango Department of Applied Microbiology, Dr. Harisingh Gour Vishwavidyalaya, Saga 470 001, India Received 10 May 2014; Revised 03 September 2014 Over the recent years glutaminase free L-asparaginase has gained more importance due to better therapeutic properties for treatment of acute lymphoblastic leukemia. Actinomycetes are known for L-asparaginase activity. In the current study, 80 actinomycetes were isolated from various soil habitats by serial dilution technique. Presence of L-asparaginase was investigated in a total of 240 actinomycetes by tubed agar method using modified M-9 medium. A total of 165 actinomycetes were found positive for L-asparaginase activity. Among these, 57 actinomycetes producing larger zones of L-asparagine hydrolysis were further screened for their capacity to produce glutaminase-free L-asparaginase. Four L-glutaminase-free actinomycetes were found to be potential L-asparaginase producers. These actinomycetes were identified as Streptomyces cyaneus (SAP 1287, CFS 1560), S. exfoliates (CFS 1557) and S. phaeochromogenes (GS 1573) on the basis of morphological and biochemical identification studies. Maximum L-asparaginase activity (19.2 Uml-1) was observed in culture filtrate of S. phaeochromogenes under submerged fermentation. Results indicate that S. phaeochromogenes could be a potential source of glutaminase free L-asparaginase for commercial purpose. To the best of our knowledge, this is the first report on production of glutaminase free L-asparaginase from S. cyaneus, S. exfoliatus and S. phaeochromogenes. Keywords: Actinobacteria, Acute lymphoblastic leukemia (ALL), L-Glutaminase, Leukemia, Streptomyces

L-asparaginase (L-asparagine amido hydrolase, EC 3.5.1.1.) is an effective therapeutic enzyme due to its use mainly in acute lymphoblastic leukemia (ALL) and lymphosarcoma1,2. L-asparaginase hydrolyzes L-asparagine, an essential nutrient for leukemic cells, into L-aspartic acid and ammonia. The leukemia cells are dependent on exogenous supply of L-asparagine for their growth and survival as they lack L-asparagine synthetase activity. However, normal cells can synthesize their own L-asparagine, and thus, remain unaffected by its rapid depletion produced by extraneous L-asparaginase. The L-asparaginase action leaves leukemic cells starved for L-asparagine which leads to inhibition of protein synthesis in leukemia cells1. Microbial L-asparaginases are basically used in clinical purposes3 . Today several commercial formulations of L-asparaginase, namely ELSPAR, Erwinase, Oncospar and PEG-L-asparaginase, etc. are available in the market for treatment of ALL 1. Most of the L-asparaginases produced from various microorganisms also possess associated L-glutaminase activity. Presence of L-glutaminase —————— *Correspondence: E-mail: [email protected]

activity in L-asparaginase preparation is reported to cause various side effects during enzyme therapy4,5 . Microbial diversity is being explored for obtaining novel sources of L-asparaginase showing less side effects for better treatment of ALL. Actinomycetes growing in various habitats have shown potential for varied applications viz., production of industrial enzymes 6-9, biosynthesis of gold nanotubes10, etc. Pereira & Kamat has extensively reviewed actinobacterial diversity and their wide applications in the fields of biotechnology and medicine including bioprospecting for vital pharmaceutical compounds and drug discovery6. Few actinomycetes viz., Bacillus aryabhattai2, Nocardia asteroids 11, Streptomyces albidoflavus12, S. griseus13, S. gulbargensis14, S. karnatakensis15 and S. venezuelae15, have been reported to produce L-asparaginase. Singh and Srivastava observed higher yield in genetic algorithm based approach rather than response surface methodology 2. However, reports on glutaminase free L-asparaginase production are limited. Here, we screened actinomycetes from local areas to identify novel isolates of high yielding L-asparaginase producers with no glutaminase activity.

SAXENA et al.: GLUTAMINASE FREE L-ASPARAGINASSE FROM ACTINOMYCETES

Materials and Methods Chemicals—L-asparagine, L-glutamine, trichloroacetic acid (TCA), bovine serum albumin (BSA) (fraction V) and Nessler’s reagent were purchased from Sigma chemical co., USA. All constituents of culture media and other chemicals were purchased from Hi-Media, India. Ammonia free water prepared by ultra water purification system (Bio Age, India) was used for all experiments. Isolation of actinomycetes— Twenty soil samples were collected from four different habitats: garden soil (GS), cultivated field soil (CFS), compost (CM), and decaying organic matter (DOM) from Sagar, Madhya Pradesh, India. All the samples were dried at room temperature (28°C) and stored for further investigation. Conventional serial dilution method was followed for isolation of actinomycetes on soil extract agar medium16. Colonies with characteristic actinomycetes morphology were picked up and streaked onto above mentioned agar medium. Pure isolates of actinomycetes were maintained on yeast extract malt extract agar slants at 4°C17. Screening for L-asparaginase production— A total of 240 actinomycetes, including 80 actinomycetes isolated during present investigation and 160 actinomycetes obtained from the microbial culture collection of Department of Applied Microbiology, Dr. Harisingh Gour Vishwavidyalaya, Sagar, M.P., India were screened for L-asparaginase enzyme. L-asparaginase producing ability of actinomycete isolates was tested by modified tubed agar method18. Modified M-9 medium stabs were used throughout screening studies using phenol red (0.005%) as a pH indicator19. A loopful of actinomycetes culture picked up from 7 days old plate was inoculated in the centre of each stab surface. The stabs were incubated at 28°C for 6 days. A set of tubes without L-asparagine was also ran as control in each case. L-asparaginase activity was measured by determination of pink zone of hydrolysis after 4 days and 6 days of incubation below the colony and categorized into 5 groups viz., excellent (4.1-5.0), good (3.1- 4.0), fair (2.1-3.0), poor (0-2.0 cm), and negative (showing no pink zone) activities. L-glutaminase production by selected actinomycetes— Selected actinomycete isolates on the basis of pink zone were further examined for the presence of glutaminase production using L-glutamine (0.5% w/v) as a nitrogen source in place of L-asparagine. L-glutamine stabs were incubated at 28ºC for 6 days to allow the growth of test

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actinomycetes and were examined regularly for pink colour formation indicating positive L-glutaminase activity below the colony. Negative results led to identification of actinomycetes that produced L-asparaginase without L-glutaminase and were categorized as isolates producing glutaminase free L-asparaginase. Identification of potential actinomycetes— Potential actinomycetes on the basis of L-asparaginase and L-glutaminase screening were further characterized according to standard protocols of International Streptomyces Project (ISP) and their identity was established using software Probabilistic Identification of Bacteria for Windows (PIBwin)17. Cultural characteristics were studied by culturing potential isolates on yeast extract malt extract agar medium. Microscopic observations were carried out using coverslip culture method20. Biochemical tests such as IMVIC, nitrate reduction, H2S production, starch hydrolysis, gelatin hydrolysis, lipolysis, pectin hydrolysis, hippurate hydrolysis, lecithinase activity, elastin degradation and xanthine degradation were also performed17. Utilization of different carbon sources (i.e., sucrose, meso-inositol, mannitol, raffinose, D-melezitose, adonitol, D-meliobiose, dextran, xylitol, arabinose, fructose, sorbitol, salicin, xylose, glucose, glycerol and galactose) was determined on basal salt agar medium17. The ability to utilize nitrogen sources such as L-cysteine, L-histidine, L-hydroxyl-proline, L-phenyl-alanine, L-valine was also studied according to Bergey’s Manual of Systematic Bacteriology, I Edition, Volume IV21. Physiological characteristics such as growth at different temperature (4-42°C), effect of NaCl concentration (2-12 %), effect of pH (4-11), effect of inhibitors and antibiotic sensitivity against four different antibiotics i.e. Neomycin (N 50), Rifampicin (R 50), Oleandomycin (O 100), and Penicillin G (10 IU) were also determined. Identification of potential actinomycete isolates was done using PIBWin software based on aforesaid features. Production of L-asparaginase in submerged fermentation— Identified actinomycetes selected on the basis of L-glutaminase screening were further grown on starch broth supplemented with L-asparagine (1% w/v) for production of L-asparaginase. The medium contained (gL-1 of distilled water) starch, 10.0; L-asparagine, 10.0; K2HPO4, 2.0; NaCl, 2.0; 1 moll-1 MgSO4.7H2O, 0.05; CaCO3, 0.02; FeSO4.7H2O, 0.01; agar 20.0 (pH 7.2).

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Erlenmeyer flasks (150 ml) containing 50 ml of starch broth were autoclaved at 15 psi for 20 min. These were inoculated with 1 ml of spore suspension (containing 107 spores/ml) harvested from culture grown on yeast extract malt extract broth on a rotary shaker (150 rpm) at 28ºC for 48 h. Flasks were then incubated at 150 rpm at 28ºC for 3 days. Subsequently, mycelium was removed by centrifugation at 5000 rpm for 15 min at 4ºC. The clear supernatant thus obtained was assayed for extracellular L-asparaginase activity. Extraction of intracellular L-asparaginase— For intracellular L-asparaginase activity, cell pellet was washed twice with distilled water. Washed cells were suspended in 5 ml of Tris-HCl buffer (0.05M, pH 8.6) and lysed using a homogenizer until a homogenous suspension was formed at 4°C. Cell debris was removed by centrifugation at 8000 rpm for 10 min at 4°C and the clear supernatant was used to assay intracellular L-asparaginase activity. Assay of L-asparaginase activity— L-asparaginase activity was detected in culture filtrates and cellular extracts following the modified method of Wriston22. The reaction mixture consisting of 900 µl of L-asparagine (1% w/v) prepared in (0.05 M) tris-HCl buffer (pH 8.6) and 100 µl of crude enzyme was incubated at 37ºC for 30 min. After the incubation period, the reaction was stopped by adding 100 µl of 1.5 M trichloroacetic acid (TCA). About 100 µl of above reaction mixture was diluted by adding 800 µl of distilled water and 100 µl of Nessler’s reagent for measuring the liberated ammonia. Optical density was measured at 450 nm using microplate reader (Bio Rad, Model No. 680, U.S.) against the blank that received TCA before addition of crude enzyme. Ammonia liberated in the reaction was quantified using standard curve prepared with ammonium sulfate and L-asparaginase activity was expressed in terms of enzyme unit (U ml-1). One enzyme unit was defined

as the amount of enzyme which liberated 1 µmol of ammonia per ml per min at 37ºC. Assay of L-glutaminase activity— The culture filtrates were also tested for L-glutaminase activity by the modified method as described by Wriston22 using L-glutamine as a substrate prepared in 0.05 M TrisHCl buffer (pH 8.6) in place of L-asparagine. Results Isolation of actinomycetes— In the present work, 80 actinomycetes were isolated which included 23 isolates from garden soil, 25 from cultivated field soil, 23 from compost and 9 from decaying organic matter on soil extract agar medium (Table 1). Screening for L-asparaginase production— Out of 240 actinomycetes tested, 165 were found positive for L-asparaginase activity (Table 2) showing formation Table 1— Occurrence of actinomycetes in samples of soil and decaying matter collected from different sites of Sagar (Madhya Pradesh, India) Habitat

Locations of sampling

Garden Soil Botany Garden, (GS) (University Campus), Sagar Girls Hostel, (University Campus), Sagar Gopalganj, Sagar Cultivated Makronia (Nursery), Sagar Field Soil Makronia (CFS) (Agricultural field), Sagar Rahatgarh, Sagar Compost Devri, Sagar (CM) Patharia, Sagar Makronia, Sagar Decaying Sabji Mandi, Katra, Sagar Organic Civil Lines, Sagar Matter Makronia, Sagar (DOM)

No. of Number of samples isolates tested 2 8 2

12

1 3 1

3 9 4

2 2 1 2 2 1 1

12 10 3 10 7 2 -

Table 2— Occurrence of L-asparaginase activity in actinomycetes isolated from different samples Number of actinomycetes Habitat Animal Feed (AF) Cultivated Field Soil (CFS) Compost (CM) Decaying Organic Matter (DOM) Garden Soil (GS) Stored Agricultural Products (SAP) Soil Near Hair Saloons (SHS) Total

Tested

L-asparaginase Positive (%)

9 58 43 32 69 16 13 240

8 40 32 20 41 13 11 165

Excellent _ 2 1 2 1 _ _ 6

Grading of L-asparaginase activity based on pink zone* Good Fair Poor 4 2 2 11 15 12 10 13 8 6 8 4 13 15 12 4 4 5 3 6 2 51 63 45

Negative 1 18 11 12 28 3 2 75

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of pink zone after hydrolyzing L-asparagine. Positive actinomycetes showed varied levels of L-asparaginase activity producing different zones of hydrolysis ranging 0.6-5.0 cm. Maximum percentage of asparaginase positive actinomycetes was observed in the samples of animal feed (88.88%), followed by soil near hair saloons (84.61%), stored agricultural products (81.25%), compost (74.41%), cultivated field soil (68.96%), decaying organic matter (62.50%) and garden soil (59.42%). However, out of these, only 6 actinomycetes (CFS 1066, CM 1101, DOM 1155, DOM 1178, CFS 1299 and GS 1509) showed excellent L-asparaginase activity. L-asparaginase activity in excellent L-asparaginase producers as seen in stabs ranged 4.2-5.0 cm. Screening for L-glutaminase production— A total of 57 actinomycetes showing excellent and good L-asparaginase activity were further screened for L-glutaminase enzyme production. About 51 of them were found positive for L-glutaminase activity; 37 fair, and 14 poor L-glutaminase producing ability. Only 4 actinomycetes isolates (SAP 1287, CFS 1557, CFS 1560 and GS 1573) did not produce any pink zone on modified M-9 medium stabs containing L-glutamine even after 6 days of incubation. Based on

these results, above 4 actinomycetes isolates were taken as L-asparaginase producers showing no glutaminase production. L-asparaginase activity in selected 4 potential actinomycete isolates as seen in tubed agar method ranged 3.2-4.0 cm. Identification of potential actinomycetes— All four glutaminase-free asparaginase producing test actinomycetes (i.e., SAP 1287, CFS 1560, CFS 1557 and GS 1573) exhibited rapid growth on yeast extract malt extract agar (YEMA) medium. The colour of substrate and spore mycelium varied from one isolate to another (Table 3). Morphology as studied using coverslip culture technique revealed that three of the isolates exhibited spiral spore chain morphology while one actinomycete i.e., CFS 1557 exhibited rectiflexible spores chain morphology (Fig.1A and B). Biochemical characteristics of potential actinomycetes are described in Table 3. None of the isolates were positive for indole production and VP test, and also were not able to utilize citrate. Two isolates of actinomycetes SAP 1287 and GS 1573 showed a strong positive methyl red test. Except test actinomycete SAP 1287, all the test actinomycetes failed to reduce nitrate to nitrite. Most of the isolates were able to hydrolyze polymeric substrates such as

Table 3— Morphological and biochemical characteristics of potential actinomycete isolates Properties Spore chain morphology (Rectiflexible/Spiral) Color of spore mass Diffusible pigment produced (Yellow/ Brown) Substrate mycelium

SAP 1287 Spiral Dark grey Light Brown Wrinkled, Whitish grey ++ -

Melanin on peptone iron agar Melanin on tyrosine agar Biochemical characteristics Indole production Methyl red ++ Vogesproskauer Citrate utilization Nitrate reduction + Hydrogen sulphide production Starch hydrolysis Gelatin hydrolysis Lipid hydrolysis +++ Pectin hydrolysis Hippurate hydrolysis Lecithinase activity Elastin degradation ++ Xanthine degradation ++ (+) Positive; (++) Strong positive; (+++) Excellent; (-) Negative

CFS 1557 Rectiflexible Rosy pink Light Brown Whitish + +

CFS 1560 Spiral White grey None Wrinkled, Yellowish White ++ -

GS 1573 Spiral Light pink Yellow Smooth, Yellowish -

+ ++ ++ + ++ + ++ ++

++ ++ +++ ++ ++

++ +++ + ++ ++ ++ + ++ ++

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INDIAN J EXP BIOL, DECEMBER 2015 Table 4— Utilization of carbon and nitrogen sources by glutaminase free L-asparaginase producing actinomycete isolates

Fig. 1—(A) Spiral spore chain of Streptomyces phaeochromogenes GS 1573 (100X); and (B) Rectiflexible spore chain of Streptomyces exfoliatus CFS 1557 (100X)

starch, gelatin, lipid and pectin. Only one isolate GS 1573 showed hippurate utilization and formation of crystals of benzoic acid in the culture medium. Most of the isolates were found positive for elastin and xanthine degradation. All the selected actinomycetes utilized carbon sources glycerol, galactose, glucose, xylose and fructose to different extent (Table 4). Most of the isolates showed poor growth and failed to utilize sucrose, raffinose, D-melezitose, adonitol, D-meliobiose, dextran, xylitol, arabinose and sorbitol as carbon source. Potential actinomycetes showed excellent growth in the presence of L-histidine. The second best nitrogen source was found to be L-hydroxyproline. All potential actinomycetes showed good growth in the presence of 0.001% (w/v) potassium tellurite and were found to be resistant against this inhibitor. Two actinomycetes, CFS 1557 and CFS 1560, also exhibited growth on phenol (0.1%). The actinomycetes isolates were found to be sensitive to sodium azide (0.01%) and thallous acetate (0.001%) and showed no growth. The assessment of physiological characteristics of the potential actinomycete isolates revealed that they could grow well at 25°C and 37°C. Optimum temperature for growth of all test actinomycetes was found to be 30°C. All test actinomycetes were found to tolerate a wide range of pH, 6-11. Optimum growth was exhibited at 2 % NaCl by all the test actinomycetes. Actinomycete isolate CFS 1560 was found sensitive against Neomycin (50 µg/ml) and showed an inhibition zone of 12 mm. Two actinomycete isolates, SAP 1287 and GS 1573 showed zone inhibition in the range of 22-33 mm against rifampicin (50 µg/ml). In the present study, potential isolates of actinomycetes were identified on the basis of spore chain morphology, pigment production, biochemical features, growth in the presence of inhibitors and sensitivity to different antibiotics, etc., using PIBWin

Carbon source utilization Sugars SAP 1287 CFS 1557 CFS 1560 GS 1573 Sucrose + + + Meso-Inositol + +++ + Mannitol + +++ + Raffinose + + + + D- Melezitose + + + + Adonitol + + D-Meliobiose + + + + Dextran + + + Xylitol + + Arabinose + + + + Fructose +++ ++ + ++ Sorbitol + + + Salicin +++ + + ++ Xylose ++ ++ + +++ Glucose +++ +++ ++ + Glycerol +++ ++ ++ +++ Galactose ++ ++ + +++ Carbon free + + Nitrogen source utilization Amino acids SAP 1287 CFS 1557 CFS 1560 GS 1573 L-cysteine + +++ + L-histidine +++ ++ +++ ++ L-hydroxyproline + ++ +++ + L-phenylalanine + + + + L-valine + + +++ + (+) Positive; (++) Strong positive; (+++) Excellent; (-) Negative

software. Two actinomycete isolates SAP 1287 and CFS 1560 have been found closely related to Streptomyces cyaneus of which ID scores ranged from 0.998 to 0.968. PIBWin ID score of one actinomycete isolate was found to be 0.999 and was identified as S. exfoliatus. Test actinomycete GS 1573 showed maximum similarity with known strains of S. phaeochromogenes with an ID score of 0.995. Production of L-asparaginase under submerged fermentation— L-asparaginase activity was measured both in culture filtrate and cellular extracts. Data given in Table 5 indicate that potential actinomycetes isolate produced L-asparaginase in range of 11.75 U ml-1 to 19.2 U ml-1 after 3 days of growth. S. phaeochromogenes GS 1573 exhibited maximum L-asparaginase activity (19.2 U ml-1) under submerged fermentation conditions. Interestingly, actinomycetes isolate S. exfoliatus CFS 1557 and S. cyaneus GS 1573 also showed intracellular L-asparaginase activity (Table 5). No L-glutaminase

SAXENA et al.: GLUTAMINASE FREE L-ASPARAGINASSE FROM ACTINOMYCETES Table 5— Extracellular and intracellular L-asparaginase activity of selected glutaminase-free actinomycetes (Culture conditions: 28°C, 72 h, 150 rpm) Test actinomycetes

Extracellular Intracellular L-asparaginase L-asparaginase activity (U/ml) activity (U/ml)

Streptomyces cyaneus 14.25±0.04 Nil (SAP 1287) Streptomyces exfoliatus 14.5±0.01 4.07±0.00 (CFS 1557) Streptomyces cyaneus 11.75±0.0 Nil (CFS 1560) Streptomyces phaeochromogenes 19.2±0.0 3.73±0.01 (GS 1573) Data are the Mean ± SD of three independent determinations

activity was detected either in culture filtrates or in intracellular extracts of all 4 potential actinomycete isolates which indicated that L-asparaginase was free from L-glutaminase activity. Discussion Side-effects caused by bacterial L-asparaginase during therapy have drawn attention towards finding novel producers of L-asparaginase. A number of research workers have reported that actinomycetes from soil possess high metabolic potential. Most of the studies on actinomycetes have focused on antibiotic/antifungal production, only few reports are on their enzymatic potential6,23-25. The need of novel metabolites from actinomycetes requires isolation and screening of large number of new isolates from unexplored areas. In the present study, maximum numbers of actinomycetes were found in cultivated field soil (CFS) samples possibly due to its nutrient richness. Garden soil and compost samples also harbored high number of actinomycetes due to availability of high amount of organic matter in these samples. Minimum number of actinomycetes was recorded from decaying organic matter. This observation suggests that actinomycetes are distributed in all habitats, however, their number vary in each habitat. Screening of L-asparaginase was envisaged to carry out a systematic evaluation of members of this group isolated from local samples. Various researchers also used similar technique for screening of L-asparaginase using phenol red dye for various microorganisms26-28. Test actinomycetes did not produce any pink zone in control i.e., M-9 media incorporated without L-asparagine. Thus, results of the present study indicate that the formation of pink

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zone is due to only L-asparaginase production in medium leading to formation of ammonia which changes pH alkaline. In the present study, 0.005% of phenol red was added into the modified M-9 medium. Thus, this concentration of phenol red in the medium keeps the colour yellow for a better identification of pink zone in the stabs. While it was observed that higher concentration of phenol red gives dark red colour which makes difficult to identify the pink zone. Screening with phenol red dye by tubed agar method revealed that presence of dye did not inhibit the growth of actinomycetes and pink zone appeared around colony. The results presented here further strengthens the finding that actinomycetes are a good source of L-asparaginase12,29,30. The use of L-asparaginase as a therapeutic enzyme implies a glutaminase free enzyme to minimize various risk factors caused during therapy31. In our studies, all the actinomycetes showed L-glutaminase activity except four isolates which were found to be highly specific for L-asparagine. Few workers have reported L-asparaginases with no glutaminase activity32,33. L-asparaginase production from Bacillus licheniformis exhibited low glutaminase activity34. Primary identification of potential test actinomycetes was done on the basis of spore chain morphology. On the basis of morphological characteristics, all the four isolates were found belonging to the genus Streptomyces showing typical Streptomyces type morphology. Several workers observed Streptomyces as the most dominant and frequent genus in any actinomycete population35. The potential actinomycete isolates were further characterized and identified as different species of Streptomyces. The present results suggest that isolates of actinomycetes varied from each other in their biochemical and physiological features. Actinomycete isolate CFS 1557 showed maximum ID score compared to other actinomycetes and identified as S. exfoliatus. Microorganisms are well recognized producers of extracellular as well as intracellular L-asparaginases. All the four test actinomycetes showed extracellular production of enzyme. S. gulbargensis showed 8.2 IU enzyme activity under shake flask conditions after 7 days of incubation while it produced 4.56 IU of L-asparaginase using groundnut cake extract as a substrate under SSF14. Sahu et al.30 reported slightly higher enzyme activity in the range of 31.2-35.6 µg ammonia/ml/h from various species of Streptomyces

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isolated from fish gut under submerged fermentation. Reports indicate many incidences of L-asparaginases being intracellular in nature12,29. Thus, all four promising test actinomycetes were also examined for intracellular L-asparaginase activity. Among these, two actinomycete isolates (CFS 1557 and GS 1573) showed presence of both intracellular and extracellular L-asparaginase. Abdel et al.36 reported intracellular and extracellular L-asparaginase from S. longsporusflavus. These findings show that all potential actinomycetes exhibited more extracellular L-asparaginase activity in comparison to intracellular activity. It was observed in our studies that S. Phaeochromogenes showed maximum L-asparaginase activity both in tubed agar method (4.0 cm) and in submerged fermentation (19.2 U ml-1). Thus, it was confirmed that there is a positive correlation between pink zone of hydrolysis formed by tubed agar method and L-asparaginase production under submerged fermentation. Glutaminase activity was not detected in extracellular or intracellular preparations of these selected isolates. This study concludes that S. phaeochromogenes is an efficient extracellular producer of L-asparaginase compared to other test actinomycetes and holds potential for treatment of acute lymphoblastic leukemia. Acknowledgement One of the authors, AS, acknowledges Madhya Pradesh Biotechnology Council, Bhopal for providing financial support during work tenure. References

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