INTRODUCTION:

L-glutaminase is omnipresent in the biological world and all organisms from bacteria to human beings possess this enzyme. L-glutaminase belongs to aminohydrolasefamily of enzymes^[1]. Aminohydrolase consists of two classes of enzymes that are involved in deamidation of glutamine. The first class includes glutaminase which has high specificity for glutamine and catalyzes the hydrolysis of glutamine to glutamic acid and ammonia^[2]. Second class includes the enzyme that has less specificity. It includes the enzyme that catalyzes hydrolysis of glutamine to glutamic acid and asparagine to aspartic acid. L-glutaminase can be used as an enzyme therapy for cancer, especially for acute lymphocytic leukemia^[3]. Mammalian tissues expressing this enzyme generate energy using L-glutamine as major respiratory fuel^[4]. Normal human cells have L-glutamine synthetase, thus they are not dependent on exogenous supply of glutamine (L-glutamine acts as an obligate nitrogen donor for purine and pyrimidines biosynthesis in living cells), while cancer cells lack this enzyme and depends on supply of L-glutamine from blood for their growth and survival.

Therefore, in the presence of L-glutaminase, L-glutamine is used up leading to the death of tumour cells^[5]. L-glutaminase also plays an important role in synthesizing the excitatory neurotransmitter glutamate, in the brain, which is the precursor for the inhibitory neurotransmitter, gamma-aminobutyric acid. Further the enzyme also has a role in regulation of glutamine and glutamate concentrations in the cerebrum, which are crucial to certain processes as ammonia detoxification^[6]. Besides L-glutaminase can also be used as an antiretroviral agent^[7], as biosensors to monitor L-glutamine level so that cancer cells can be detected^[8]. In food industry L-glutaminase is becoming increasingly important for enhancing the flavour of fermented foods such as soy sauce and other food products by increasing the glutamic acid content, and also the aroma of food products^[9]. It also causes deamidation of several proteins including alpha-lactalbumin, alpha-zein, wheat gluten, and skim milk. Deamidation causes change in functional properties that is solubility, foaming capacity and emulsification properties of food proteins^[10].

Microorganisms such as bacteria, yeast, fungi are potential sources for L-glutaminase, and using these microbes for L-glutaminase production is more preferable as they have simple growth requirements, easy to process and has cheaper production costs. L-glutaminase is also produced by Actinomycetes; a diverse group, possessing mycelial growth. They are gram-positive, aerobic eubacteria possessing asexual spores. They contain many genera distinguished by combination of structural and chemical properties. Actinobacteria is a phylum under actinomycetes characterized by high G+C content^[11]. The natural occurrence of actinobacteria is mostly restricted to soil but in recent years many actinobacteria have been isolated from marine environments which are capable of producing biologically active compounds^[4].

The marine environment is considerably different from the terrestrial environment in context to extreme parameters like salinity, less amount of light and hydrostatic pressure. Marine environment is virtually untapped and also a good source for novel actinobacterial diversity. The distribution of marine actinobacteria is largely unexplored and the presence of indigenous marine actinobacteria in ocean remains elusive. Marine actinobacteria has a diverse range of enzyme activities and are capable of catalyzing various biochemical reactions. The saline nature of marine water resembles human blood plasma and hence can be used for different therapeutic purposes without having any side effects to humans. Thus, marine environment is a unique source for providing salt tolerant and thermostable L-glutaminase. Therefore we narrow down our study to screening of L-glutaminase producing marine actinobacteria as less has been investigated about this^[12].

Anti-oxidants have the ability to scavenge and inhibit free radicals. Free radicals are able to oxidize nucleic acids, proteins, lipids and therefore can initiate degenerative diseases. Biological systems have highly reactive free radicals and oxygen species. Antioxidants have wide applications as dietary supplements and can prevent diseases like cancer, coronary heart disease and even altitude sickness^[13-15]. Antioxidants also have numerous industrial uses, as food preservatives, in cosmetics and to prevent the degradation of rubber and gasoline^[16].

Hence, we concentrate on identification of a novel marine actinobacteria capable of producing the enzyme L-glutaminase and application of L-glutaminase as an anti-oxidant. With this view, the current study focuses on screening of L-glutaminase producing marine actinobacteria and its anti-oxidant and haemolytic activities.

MATERIALS AND METHODS:

Sample collection

Soil samples were collected from salt pan soil in Kothapattanam, Ongole, Andhra Pradesh (15° 30’ 0” N, 80° 3’ 0” E), India during December 2013. Soil samples were collected at a depth of 10-25cm. The collected sample were kept in a sterilized container, then transferred to the laboratory and stored at 4°C for further use^[17].

Isolation of Marine Actinobacteria

Isolation of actinobacteria was performed by using selective media such as Actinomycetes Isolation Agar (AIA), Kuster’s agar and Starch Caesin agar. All the media were supplemented with a final concentration of 100 µg/l cyclohexamide and 15 µg/l nalidixic acid to inhibit fungal and bacterial growth. A 10 fold serial dilution series was made and plated in triplicate on agar plates. The plates were incubated at room temperature (28°C) and monitored periodically over 3 months for actinobacterial growth.

Primary screening of Actinobacteria

For primary screening, 12 isolates were selected on the basis of powdery and leathery growth of actinomycetes on actinomycetes isolation agar. The isolated actinobacterial colonies were inoculated on Minimal glutamine agar plates for detecting L-glutaminase producing actinomycetes. Minimal glutamine agar contained (g/l) 0.5 KCl, 1 KH₂PO₄, 0.5MgSO₄, 0.1 ZnSO₄, 25 NaCl, 10 L-glutamine and 0.012 of 2.5% phenol red ^[18].

Secondary Screening of Actinobacteria

Four isolates showing pink coloured colonies on minimal glutamine agar plates were selected. The isolates were inoculated in media containing 5g glucose, 5g Na₂HPO₄, 3g KH₂PO₄, 0.49g CaCl₂, and 2.5%w/v ethanolic phenol red solution 0.06 ml with pH adjusted to 7.6. Controls were kept where the same cultures were grown in media containing (a) 5g NaNO₃ instead of glutamine, (b) media containing glutamine as the carbon source and (c) same media without dye. The isolated colonies were grown in 100ml flask with 25ml of media kept in shaker at 180rpm at 27°C for 48 h. Cultures that showed utilization and production of glutamine is indicated by decrease in pH and then taken up for further analysis of glutaminase ^[18].

L-glutaminase Assay:

This was done by direct Nesslerization ^[19]. Enzymatic reaction mixture contains 1ml 0f 1% L-glutamine in citrate buffer and 1ml of crude enzyme incubated at 30°C for 1 h. This enzymatic assay was stopped by adding 1.5M trichloroacetic acid (0.5ml). Reaction mixture was centrifuged at 5000rpm for 5min. The released ammonium was determined by using 0.5ml Nessler’s reagent. After 15minutes the developed colour was measured at 480nm using spectrophotometer. Blanks of substrate and enzyme were used as controls. The ammonium concentration was determined from standard curve of ammonium sulphate. One unit of L-glutaminase is the amount of enzyme that liberates 1µ mol of ammonia^[20].

Antioxidant assay

Based on the results obtained from the secondary screening, four isolates were selected based on L-glutaminase production and anti-oxidant assays were performed.

DPPH radical scavenging activity

Cultures were centrifuged at 5000rpm for 10minutes. Supernatants were collected for anti-oxidant assay. In test tubes, 1ml of supernatant from each strain was taken. Then 2ml DPPH reagent (2, 2-diphenyl-1-picrylhydrazyl) was added to the supernatant in dark. DPPH is a free radical and it is violet in colour. Test tubes were incubated for 20minutes at room temperature in dark condition. The degree of reduction of the reagent was measured as absorbance in UV–vis spectrophotometer at 517nm ^[21].

Percentage of scavenging activity was calculated as:

% Scavenging rate= (Control-Absorbance of test/Control)*100

Reducing power Assay

Reducing power was measured using the protocol ^{[22, 23]} given as follows. In this 1.0ml of the sample was dissolved in 1.0ml of phosphate buffer and was mixed well with 1.0ml of 1% potassium ferricyanide [K₃Fe(CN)₆] and then this mixture was incubated at 50^oC for 20minutes during which ferricyanide is reduced to ferrocyanide. Subsequently 1.0ml of trichloroacetic acid, and 1.0milliq distilled water was added. Finally the solution was mixed with 0.1ml of 0.1% ferric chloride. Mixture was incubated at 50^oC for 10minutes. Absorbance was measured at 700nm.

Metal chelating assay

The chelating activity was measured using the following protocol ^[24]. In this method 0.5ml of each extract were mixed with 1.6milliq of distilled water. Then 0.05ml of FeCl₂ was added in each test tube. This reaction was initiated by the addition of 0.1ml of 5Mm ferrozine. The solution was mixed well and allowed to stand for 10 minutes at 40°C. After incubation period, absorbance was measured spectrophotometrically at 562nm. Distilled water was used as control. Instead of FeCl₂ distilled water was used as blank. Ferrous ion-chelating ability was calculated as follows:

%scavenging activity

(Ferrous ion chelating ability)= [1- (A₁-A₂)/A₀] * 100

Where, A₀ is absorbance of control, A₁ is reaction mixture; A₂is absorbance without FeCl_2.

Haemolytic activity

The haemolytic activity of L-glutaminase producing actinobacteria was measured using the following protocol ^[25]. In this assay 5ml of blood containing heparin was centrifuged at 15000rpm for 30minutes. Supernatant containing plasma was discarded and pellet containing red blood cells was washed with 0.75% saline by centrifugation at 1500rpm for 5minutes. The cells were suspended in normal saline. 0.5ml of cell suspension was then mixed with sample. The mixture was incubated for 30minutes at 37°C and then centrifuged at 1500rpm for 10 minutes. The free haemoglobin in supernatant was measured using UV-Vis spectrophotometer at 540nm. Distilled water and phosphate buffer saline was used as minimal and maximal haemolytic controls. The activity was calculated as follows-

%Haemolytic activity = (A_t- A_n) / (A_c- A_n) * 100

Where A_tis absorbance of sample, A_nis absorbance of control and A_c absorbance of control

Taxonomic characterization of the potential strain

The efficacious actinobacteria are characterized by morphological and biochemical methods and the results were compared with Nonomura key 1974, Shirling and Gottlieb 1966 and with Bergey’s manual of Determinative Bacteriology^[26].

Morphological characteristics

Different actinobacterial isolates were streaked on seven different international Streptomyces Project media (ISP 1 to ISP 7) and kept for incubation for 7 days at room temperature. Different morphological characters were observed. Based on the morphological characters the isolate was confirmed as actinomycetes. Later on the Starch Casein Agar medium (SCA), the isolated strain was streaked, and by coverslip method the morphology of the colony was observed under microscope.

Biochemical Characterization

The ability of isolated actinobacteria to utilise different sugars were tested. The sugars used were mannitol, mannose, maltose, xylose, sucrose, lactose by International Streptomyces Project (ISP). These carbon molecules were then sterilised by ether sterilisation^[27].

Generic investigation

The L-glutaminase producing actinobacteria was identified using by cell wall composition analysis (amino acids and whole cell sugars analysis)^[28].

RESULTS AND DISCUSSION:

Isolation of Marine actinobacteria

From the Actinomycetes isolation agar plates colonies showing powdery or leathery growth and specific microscopic observations like gram-positive character, non-motile character were taken for primary screening. Out of 20 collected marine samples, 12 isolates were identified to be actinobacteria. In most of the microbial screening methods used to check the enzyme production, first step includes isolation of microbes from certain environments and then screening of enzyme activity by observing the zone of clearance on agar media supplemented with suitable substrates ^[18].

Primary Screening

As reported in^[29], 25 isolates were screened for L-glutaminase activity and out of that 1 exhibited positive activity; also in ^[30] modified Czapek Dox media for isolation of L-glutaminase producing actinobacteria 10 isolates showed positive result out of 50 isolates. However, in this paper using minimal glutamine agar, out of the 12 isolated actinobacterial isolates 4 isolates showed positive result by production of pink colonies. In minimal glutamine agar, L-glutamine was the only nitrogen and carbon source, thus the organisms capable of producing L- glutaminase would be able to grow in this media. The minimal glutamine agar media contains phenol red as indicator of pH. The pH indicator shows colour change from yellow to pink indicating glutaminase production. The four actinobacterial isolates showed positive utilization of L-glutamine present in the media resulting in the production of L-glutamic acid which changed the indicator’s colour from yellow to pink. Thus the use of selective media and presence of specific indicator makes the media suitable for direct and selective isolation of L-glutaminase producing marine actinobacteria. As compared to the above mentioned results reported earlier, in this study maximum numbers of colonies were obtained on Minimal glutamine agar media.

Fig. 1 Minimal glutamine agar plate displaying the growth of L-glutaminase producing isolates

Secondary Screening

On the basis of the results obtained on minimal glutamine agar plates, the isolates were inoculated in broth. 4 flasks containing the glutamine media showed decreased in pH. These flasks were selected for further analysis. The culture broth was centrifuged at 5000rpm for 10 minutes and supernatant was used as crude enzyme for the assay.

L- glutaminase assay:

This assay was carried out for the above 4 isolates. The principle behind this assay is that the L-glutaminase producing isolates catalyses the hydrolysis of L-glutamine to ammonia and L- glutamic acid. This released ammonia is detected by reaction with nessler’s reagent. It turns the pale solution of nessler’s reagent to deeper yellow. At higher concentration it even forms brown precipitate. Thus, determination of released ammonia concentration is an indirect indication of the amount of L-glutaminase produced by the isolates showing positive result.

Isolates	Enzyme activity (IU/ml)
BSAIP 1	0.27
BSAIP 5	1.77
BSAIP 7	1.33
BSAIP 8	0.16

Table 1.1 L-glutaminase activity of the actinomycetes isolates

From Table 1.1 it is clear that the isolates produce L-glutaminase but its concentration is less as compared to the results that have been reported earlier ^[29,4]. In ^[4]media optimization was done using Response surface methodology which yielded L-glutaminase activity of 71.23 Units and in ^[26]optimization of culture conditions were done and the maximum L-glutaminase production obtained was 14.5IU/ml. However in present study different parameters for increasing the concentration of L-glutaminase were not used and thus by analyzing one factor at a time the production of L-glutaminase can be increased.

Antioxidant Assay

DPPH radical scavenging activity

2, 2-dipenyl-1-picrylhydrazyl (DPPH) is a biological free radical and it is violet in colour.

The magnitude of anti-oxidation ability of sample can be expressed by their capacity to scavenge DPPH radical. Based on this property, antioxidants present in the sample will turn the free radical into yellow colour. This change of colour from violet to yellow thus indicates a positive test^[8].

From the obtained results it is concluded that strain BSAIP5 has highest free radical scavenging activity compared to the others. The above strain BSAIP5 shows the highest percentage of scavenging activity, it is higher than the value reported in ^[29^].

Fig. 2 Scavenging activity of different L-glutaminase producing isolates

Reducing Power Assay

The electron donating capability of an antioxidant is measured by the reducing power assay. When reducers are present they convert Fe³⁺/ ferricyanide complex to the ferrous form, which is a significant indicator of antioxidant activity. Green colour indicates positive test. Increase in the absorbance of the reaction mixtures indicates increase in reducing power. Strain BSAIP 5 showed maximum reducing power.

Fig. 3

Metal Chelating Assay

Chelating property of the transition metals prevents catalysis of hydrogen peroxide decomposition. If chelating agents are present, the complex formation is inhibited and there is reduction in the red colour. Chelating activity of sample is determined by measurement of colour. The transition metal ion Fe2+ has the ability to move electron, by this it can allow propagation and formation of radical reaction. Strain BSAIP 5 showed highest metal chelating activity.

Fig. 4 Metal chelating activity of L-glutaminase producing isolates

Haemolytic assay

In vitro haemolytic assay using spectroscopic methods provides an effective and easy method for the quantitative measurement of heamolyte. This method helps to evaluate the effect of different concentration on biomolecules of the human erythrocytes.

No haemolytic activity was observed, indicating that the L-glutaminase producing isolates are not toxic to the human red blood cells.

Characterization of potential strain

The strain BSAIP5 contains LL-Diaminopilimic acid and it also contains Glycine in its cell wall. Presence of both of this compound indicates that the cell wall belongs to chemotype- I, which is characteristic of the genera Streptomyces, Streptoverticillium, Chainia, Actinopycnidium, Actinosporangium, Elyptrosporangium, Microellobosporia. The morphological characters of strain BSAIP5 are similar to the genus Streptomyces.

Table1.2: Morphological and Biochemical Characterization of BSAIP5 and S. humidus:

Characteristics	BSAIP5	*S. humidus*
Morphological characteristics
Colour of aerial mycelium	Grey	Grey
Melanoid pigment	Nil	Nil
Reverse side pigment	Nil	Nil
Soluble pigment	Nil	Nil
Spore chain morphology	Spirales	Spirales
Spore surface morphology	Smooth	Smooth
Utilisation of sole carbon sources
Sucrose	+	+
Dextrose	+	+
Maltose	+	+
Mannose	++	++
Xylose	++	++
Mannitol	++	++

The morphological and biochemical results of L-glutaminase producing strain BSAIP5 are compared, with those of Streptomyces species given in the key of Nonomura and also with the species described in the Bergey’s Manual of Determinative Bacteriology. The strain BSAIP5 showed resembling characters to the reference strain S. humidus. Hence the BSAIP5 has been tentatively identified S. humidus.

Table1.3: Generic investigation

Strain no

LL-DAP

Meso-DAP

Glycine

Whole cell

sugars

Wall type

S. humidus

BSAIP5

CONCLUSION:

In this study, screening of Actinomycetes was done by using samples collected from Kothapattanam, Ongole, Andhra Pradesh (15° 30’ 0”N, 80° 3’ 0” E), India. From this, four L-glutaminase producing isolates were isolated. Further, with these L-glutaminase producing isolates different anti-oxidant assays were performed. The isolates also showed anti-oxidant properties like scavenging of free radical, reducing power and metal chelating capability. No haemolytic activity was seen. Thus, these 4 isolated strains were found to be potential producers of L-glutaminase. Strain BSAIP5 showed highest enzyme production and anti- oxidant activities. Future prospects include designing a protocol to increase the enzyme activity of L-glutaminase.

ACKNOWLEDGEMENT:

The authors are very much grateful to the management and staff of VIT University Vellore, TN, India for supporting this study.

REFERENCES:

1. Bulbul D, Karakus E. Production and optimization of L-glutaminase enzyme from Hypocrea jecornia pure culture. Preparative Biochemistry and Biotechnology. 43(4); 2013: 385-97.

2. Archibald, R. M. Preparation and assay of glutaminase for glutamine determinations. Journal of Biological Chemistry. 2013: 657-667.

3. P. Singh and R.M. Banik. Biochemical Characterization and Anti-Tumor study of L-glutaminase from Bacillus cereus MTCC 1305. Applied Biochemistry and Biotechnology. 171(2); 2013: 522-31.

4. Padma.V.Iyer and Rekha S. Singhal. Screening and Selection of Marine Isolate for L-Glutaminase Production and Media Optimization Using Response Surface Methodology. Applied Biochemistry and Biotechnology. 159; 2009: 233-250.

5. Ashraf S.A.El-Sayed.L-glutaminase production by Trichoderma koningii under solid-state fermentation. Indian Journal of Microbiology.49; 2009: 243-250.

6. Olalla, L., Aledo, C., Bannenberg, G. and Marquez C. The c-terminus of human glutaminase L mediates association with PDZ domain-containing proteins. FEBS Letters. 488; 2001: 116–122.

7. Roberts J and McGregor WG. Inhibition of mouse retroviral disease by bioactive glutaminase- asparaginase. Journal of General Virology. 72(2); 1991: 299–305.

8. Sabu A, Chandrasekaran M and Pandey A. Biopotential of microbial glutaminases. Chemistry Today. 18; 2000: 21–25.

9. Ito K, Koyama Y, Hanya Y. Identification of glutaminase genes of Aspergilllus sojae involved in glutamate production during soy sauce fermentation. Bioscience, Biotechnology, and Biochemistry. 77(9); 2013: 1832-40.

10. Inthawoot Suppavorasatit. Deamidation of soil protein by protein glutaminase: process evaluation and effect of deamidation on protein functional properties and flavour protein interactions. University of Illinois. 2012.

11. Arulappan Jayaprakash Priya et al. Detection of Antimicrobial and Antioxidant activities in Marine Actinobacteria isolated from Puducherry coastal regions. Journal of Modern Biotechnology. 2012.

12. Tanti Yulianti, Ekowati Chasanah, and Usman Sumo Friend Tambunan. Screening and Characterization of L-glutaminase Produced by Bacteria Isolated from Sangihe Talaud Sea. (unpublished).

13. Ito N. and Hirose, M. Antioxidants .carcinogenic and chemopreventive properties. Advances in Cancer Research. 53; 1989: 247.

14. Gerber, M. Astre, C., Segala, C., Samtot, M., Scali, J and Lafontaine. Oxidant – antioxidant status alterations in cancer patients: relationship to tumor progression. Journal of Nutrition.126 (4); 1996: 1201s.

15. Kris- ertherton, P.M., Harris, W.S. Fish consumption, fish oil, omega-3-fatty acids and cardiovascular disease. Appel, L.J.2002. Circulation.106; 2012: 257-274.

16. Pandey, A. Solid-state fermentation. Biochemical Engineering Journal. 13; 2003: 81-84.

17. Sathish Kumar SR, Kokati Venkata Bhaskara Rao. In-vitro antimicrobial activity of marine actinobacteria against multidrug resistance Staphylococcus aureus. Asian Pacific Journal of Tropical Biomedicine. 2012: 787-792.

18. R. Balagurunathan, M. Radhakrishnan, S.T. Somasundaram. L-glutaminase Producing Actinomycetes from Salt pan soil–Selective Isolation, Semi Quantitative Assay and Characterization of Potential Strain. Australian Journal of Basic and Applied Sciences. 4(5); 2010: 698-705.

19. Imada A, Igarasi S, Nakahama K and Isono M (1973) Asparaginase and glutaminase activities of microorganisms. Journal of General Microbiology. 76; 1973: 85–99.

20. K. Nathia, Suraj S. Nath, J. AngayarKanni and M. Palaniswamy. In vitro Cytotoxicity of L-glutaminase against MCF cell lines. Asian Journal of Pharmaceutical and Clinical Research. 5; 2012: 171-173

21. K. Nathia, Suraj S. Nath, J. AngayarKanni and M. Palaniswamy. Screening of a high glutaminolytic enzyme producing strain and its extracellular production by solid state fermentation. International journal of pherma and bio space sciences. 3; 2011: B 297-302.

22. Daljit Singh Arora and Priyanka Chandra. February 2011. Antioxidant Activity of Aspergillus fumigatus. Microbial Technology Laboratory. International Scholarly Research Network. 2011.

23. L.W Chang, W.J Yen, S.C Huang, P.D Duh. Antioxidant activity of sesame coat. Food Chemistry. 78; 2006: 347-354.

24. G.R Zhao, Z.J Xing, T.X. Ye, Y.J.Yuan, Z.X.Guo. Antioxidant activity of Salvia miltiorrhiza and Panax notoginseng. Food Chemistry. 99; 2006: 767-774.

25. Gaurav Kumar, Loganathan Karthik and Kokati Venkata Bhaskara Rao. Haemolytic activity of Indian medicinal plants toward human erythrocytes: an in vitro study. Applied Botany. 40; 2011: 5534-5537.

26. Cross T. Growth and examination of actinomycetes some guidelines. In Bergey’s Manual of Systematic Bacteriology. Williams and Wilkins Company, Baltimore. 4; 1989: 2340-2343.

27. Shirling EB, Gottlieb D. Methods for characterization of Streptomyces sp. International Journal of Systemic Bacteriology. 16; 1996: 313-340.

28. Karthik L, Gaurav Kumar, Bhaskara Rao KV. Mutational effects on the protease producing marine actinomycetes isolated from Scylla serrata. Pharmacology online. 1; 2010: 221-227.

29. S. Krishnakumar, R. Alexis Rajan and S. Ravikumar. Extracellular production of L-glutaminase by marine alkalophilic Streptomyces sp-SBU1isolated from Cape Comorin coast. Indian Journal of Geomarine Science. 2011: 717-721.

30. N.Sajitha, S.Vasuki, M.Suja, G.Kokilam, and M. Gopinath. Screening of L-glutaminase from Sea Weed Endophytic Fungi. International Research Journal of Pharmaceutical and Applied Sciences. 2013: 206-209.

Received on 04.03.2014 Modified on 01.04.2014

Research J. Pharm. and Tech. 7(5): May, 2014; Page 544-549