INTRODUCTION:

The enzyme L-glutaminase is ubiquitous in nature and also known as an amidohydrolase enzyme^[1]. The L-glutaminase enzymes have a high specificity for glutamine and it acts as a catalyst for the conversion of L-glutamine to glutamic acid and ammonia by glutamine hydrolysis^[2,3]. The importance of the amidohydrolases aroused with the exploration of their anti-tumor properties and the studies on L-glutaminase had increased on a large scale^[4,5,6]. This enzyme is favorable for the treatment of lymphocytic leukemia. It displays anti-cancer effects by removing the L-glutamine from all of the cancerous cells^[7].

The proliferating cancerous cells avidly use up all of the L-glutamine for energy requisite and proliferation in comparison to the normal cells^[8]. Similarly, L-glutaminase also acts as a bio-sensing factor in controlling the level of glutamine in hybridoma and mammalian cell lines^[9]. It is also used as a therapeutic agent against HIV virus^[10]. A coextensive interest on large scale L-glutaminase production has aroused due to its usage in food flavoring, mainly in soya sauce for the preparation of high value chemicals like threonine^[11]. Monosodium Glutamate was prominently used as a taste enhancer in Chinese food^[10,12]. The usage has given rise to various allergies and diseases hence L-glutaminase has currently replaced it^[13,14]. Various types of microorganisms mainly bacteria, actinobacteria, fungi and yeasts are the main sources of producing L-glutaminase enzyme.

Yeasts are unicellular eukaryotes classified under the kingdom Fungi^[15]. Yeasts are prominently used in Food and Brewers industry^[16]. Marine yeasts are omnipresent almost in all areas of the aquatic ambiance^[17]. Marine yeasts are classified as innate to marine habitats^[18]. No actual physiological discoveries have been figured out to state why marine yeasts could survive in these special environmental conditions^[19]. Yeast community have been found to be decreased in population with increase in distance from land and some of the yeast species that were obtained from seawater was found in maximal quantities from the areas of high population^[20,21]. The characteristic for only salinity tolerance does not differentiate marine yeasts from terrestrial yeasts^[22]. Almost every species of yeasts can thrive in salt concentration that is present in the marine environment ^[23]. The dominant genera in marine environment are Candida sp. and Rhodotorula sp.^[20].

The marine yeasts are capable to produce bioactive compounds like amino acids, glutathione, toxins, glucans and vitamins. In addition to this, they play a peculiar role in the food and pharma industry, cosmetic, chemical industries and environmental protection^[24,25]. A large diversity of microorganisms like actinobacteria, yeasts and fungi has been found to produce L-glutaminase enzyme in large scale^[6]. L-glutaminase synthesis was also reported to be produced by various marine yeasts such as Candida and Rhodotorula species^[26].

Free radicals are basic to the biochemical and biological procedures and they are also an important part of the metabolism and all the aerobic life-forms^[27]. But these free radicals can cause various life-threatening diseases like cancer, cardiovascular diseases, Parkinson’s disease, Alzheimer’s disease and neural diseases^[28]. So, the antioxidants are having the ability to scavenge and remove the free radicals. Antioxidants are having varied applications in medical field, different industries like food and cosmetics industries, degradation of rubber and gasoline etc.^[3].

In our present study, marine yeasts were isolated from marine waters and screened for the production of L-glutaminase enzyme and also carried out the optimization and in vitro antioxidant activity of L-glutaminase enzyme.

MATERIALS AND METHODOLOGY:

Chemicals used:

All the chemicals used in this study were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai and Sisco Research Laboratories Pvt. Ltd (SRL), Mumbai, India. The chemicals were of analytical grade.

Sample collection:

Marine water samples were collected from the backwaters of Andaman and Nicobar Islands, India. The water samples were taken at the depth of 30-35cm down at sterile containers. The collected samples were brought to the Marine Biotechnology laboratory, Vellore Institute of Technology, Vellore and kept at 4ºC for further use.

Isolation of L-glutaminase producing marine yeasts:

Marine yeasts were isolated by serial dilution and spread plate technique. The samples were serially diluted up to 10^-6 dilutions. The medium, Yeast Malt agar (YMA) was prepared using marine and distilled water in 1:1 ratio and the medium was supplemented with chloramphenicol antibiotic (100mg/ml) after sterilization. About 0.1ml of aliquot were taken from all the tubes and uniformly distributed on sterile YMA medium with the help of sterile glass spreaders. The plates were incubated at 28°C for 3-5 days^[29]. The results were observed from the second day of incubation.

Characterization of marine yeast:

After 3-5 days of incubation, yeast isolates with divergent morphological features which showed creamy white or any pigmented colonies were identified. The selected colonies were sub-cultured by streaking on yeast malt agar medium and incubated at 28^oC for 3 days. The pure marine yeast isolates were characterized microscopically using gram staining and Scanning Electron Microscopy (SEM).

Screening of marine yeasts for L-glutaminase production:

a) Primary screening:

For the primary screening of L-glutaminase enzyme, the marine yeast isolates were streaked using line streaking method on minimal glutamine agar (MGA) containing (g/L) 0.5 KCL, 1 KH₂PO₄, 0.1 ZnSO_4,0.1 MgSO_4,25 NaCl, 10 L-glutamine and as indicator 0.012 of 2.5% phenol red^[30]. Yeast isolates producing L-glutaminase enzyme will result in changing the medium color from yellow to pink.

b) Secondary screening:

L-glutaminase quantitative assay was done by nesslerization method described by Imada et al.^[31]. A total of 100ml production media was prepared with the compositions (g/L) 0.25 peptone, 1.165 maltose, 0.15 yeast extract, 1 beef extract, 0.5 ammonium acetate, 0.25 glucose, 0.02 dihydrogen potassium phosphate, 0.02 sodium sulphate, 0.02 magnesium chloride, 0.0005 L-glutamine and 0.000125 phenol red. L-glutamine was added as nitrogen and carbon source. Phenol-red acts as pH indicator. The production medium (pH 7.6) was inoculated with the yeasts isolates and incubated at 28°C on rotary shaker at 125 rpm for 72 h. Potential marine yeast isolates that showed production of L-glutaminase was indicated by decrease in pH^[31]. The crude extract along with trichloroacetic acid was centrifuged at 5000 rpm^[32]. The supernatant was collected and to it 0.5mL of Nessler’s reagent was mixed to determine the amount of released ammonia. The mixture was incubated for 15 minutes and optical density was examined at 480nm. Blank and control was run simultaneously. A standard curve of ammonium sulphate was prepared to check the ammonium concentration. One unit of L-glutaminase is the amount of enzyme that can liberate 1 µmol of ammonia.

Optimization for production of L-glutaminase:

Medium optimization for the production of the enzyme L-glutaminase was done to check better production of the enzyme. The parameters optimized were different pH (2-12), temperature (0°C, 4°C, 20°C, 28°C, 37°C and 45°C), carbon source (dextrose, lactose, starch, sucrose, mannitol, and arabinose) and also the nitrogen source (peptone, casein, ammonium nitrate, ammonium sulphate, potassium nitrate and sodium nitrate)^[33].

Antioxidant activity:

DPPH scavenging activity:

Based on the scavenging capacity of the test samples, violet colour of the free radical DPPH (2,2-Diphenyl-1-picrylhydrazyl) will be change into yellow (Sabu et al., 2000). The aliquots of concentration 20, 40, 60, 80 and 100 (mg/ml) were prepared for the crude compounds and to it 2 ml of DPPH reagent (free radical) was added. The tubes were kept in dark for 20 minutes and absorbance was measured at 517nm in a spectrophotometer. For standard curve, ascorbic acid was used. Control was prepared by mixing 2 ml of methanol with 1 ml of DPPH. Methanol was used as blank^[34].

Formula for calculation of DPPH scavenging activity:

(Abs. of standard) - (Abs. of sample) / (Abs. of standard) X 100

Molecular identification of potential yeast isolate using 18s rRNA sequencing:

The potential marine yeast isolate was identified using 18s rRNA sequencing method. For extraction of genomic DNA, the yeast isolate was inoculated in previously mentioned production media and incubated at 28°C for 3 days. After the incubation period, yeast cells were separated by using centrifuge (7168×g for 10 min). The cells were then transferred in a 2 mL microcentrifuge tube and 800µl of extraction buffer (10mM EDTA pH 8, 0.1 M Tris-HCL pH 8, 3.5% CTAB, 2.5 M NaCl, 150µl of 20mg/ml proteinase K) was added along with sterilized glass beads (0.5-1 mm) and mixed properly. Then the sample was placed in 65°C for 30 min followed by centrifugation at 11,200×g for 10 min. The supernatant was collected and mixed with phenol-chloroform-isoamyl alcohol (25:24:1) in 1:1 ratio and centrifuged at 11,200×g for 10 min. Again the supernatant was collected and mixed with same amount of chloroform-isoamyl alcohol (24:1). Then the supernatant was collected and equal volume of ice-cold isopropanol was mixed with it and the mixture was incubated at -20°C for 1 h. After incubation, DNA was extracted by centrifugation at 18928×g for 15 min and the extracted DNA pellet was washed with 800µl of 70% ethanol. DNA pellet was then dissolved in TE buffer (10mM Tris-HCL pH 8, 1 mM EDTA). RNase A (20mg/ml) was added to DNA samples and incubated at 37°C for 1 h. Finally, DNA was recovered and air dried^[35]. The PCR product was amplified bi-directionally using the forward (5'-AGAGTRTGATCMTYGCTWAC-3') and reverse (5'-CGYTAMCTTWTTACGRCT-3') primers. The sequence was analyzed using ABI3730XL capillary DNA sequencer (ABI Prism 310 Genetic Analyzer, Tokyo, Japan) at Acme Progen Biotech (India) Private Limited, Salem, Tamil Nadu, India.

Statistical analysis:

All of the experiments including the optimization studies are statistically analysed using one-way ANOVA. The graphs for the experiments have been prepared in Mean ± Standard deviation format.

RESULTS AND DISCUSSION:

Isolation of marine yeast:

A total of 26 isolates were obtained from the backwater samples on yeast-malt agar (YMA) medium. The populations of yeast isolated in the present study are slightly higher than the recent report by Raj et al. where 20 marine yeasts isolates were isolated from marine sediments of Bay of Bengal, Bakkhali Coast, West Bengal, India using YMA medium^[34].

Screening of L-glutaminase producing marine yeasts:

Primary screening:

Among the recovered marine yeast isolates, morphologically distinct yeast colonies were selected for primary screening for the production of L-glutaminase enzyme. Out of them, 4 isolates DAMB1, DAMB2, DAMB3 and DAMB4 showed positive enzyme activity. In the given media L-glutamine was the only point of supply for carbon and nitrogen. The isolates having the efficiency to produce L-glutaminase only could flourish in this media. The pH indicator phenol-red showed the color change from yellow to pink indicating L-glutaminase yield as the glutamic acid produced from this reaction decreased the medium pH^[36]. Thus the use of minimal glutaminase agar was suitable for isolation of L-glutaminase producing marine yeast. The zone of hydrolysis was measured and the highest zones were found to be in DAMB1 (40mm) and DAMB3 (30mm) (Fig. 1). The zones are the confirmation for the L-glutaminase enzyme production by the yeast isolates that were been tested. In another study with seaweed endophytic fungi, out of 50 fungal isolates, 20 showed positive L-glutaminase activity^[37] whereas in our study, out of 26 yeast isolates only 4 isolates showed good enzyme activity.

a b

Fig. 1: Minimal glutamine agar (MGA) plates showing L-glutaminase enzyme producing yeast isolates

Secondary screening:

Two yeast isolates DAMB1 and DAMB3 which were showing good enzyme activity were taken for secondary screening i.e. quantitative analysis of L-glutaminase enzyme production using UV spectrophotometry. Decrease in pH was noted and revealed that the yeast isolates producing L-glutaminase also can catalyse hydrolysis of L-glutamic acid and ammonia. The ammonia produced in the flask by the isolates detected by Nessler’s reagent which turns pale solution to deep yellow. DAMB1 showed highest enzyme activity of 67% whereas DAMB3 showed enzyme activity of 53% (Fig. 2). Halotolerant yeast Zygosaccharomyces rouxii used rice bran and sesame oil cake to produce L-glutaminase enzyme^[16]. In a previous study using marine actinobacteria, the highest enzyme activity was found to be 1.77 IU/mL^[3]. The production of L-glutaminase from Streptomyces sp. was seen to be on an average of 8.029U/mL^[38].

Fig. 2: Marine yeast isolates showing L-glutaminase enzyme activity

Characterization of the potential marine yeast:

Microscopical analysis:

Fig. 3: Gram’s staining of yeast isolate DAMB1 (Magnification 100X)

Gram staining (Fig. 3) and Scanning Electron Microscopy (SEM) analysis (Fig. 4) were done to observe the microscopic characterization of the potential marine yeast isolate DAMB1.

Fig. 4: Scanning Electron Microscopy (SEM) showing yeast cell buds at magnification 6.00 K X

Medium optimization for better production of L-glutaminase enzyme:

The medium was optimized for better L-glutaminase enzyme production by varying different environmental parameters like temperature, pH, carbon sources and nitrogen sources. The isolate DAMB1 was selected for the further studies as it produced the highest amount of enzyme. The change in pH is observed due to the growth of microbial isolates and formation of other metabolic products. The changes in pH indicated the better enzyme production which was maximal at pH 8.0 (73%). The decrease in pH indicated the low microbial growth and low enzyme production (Fig. 5a). The yeast isolates were incubated at different temperature and the activity of L-glutaminase was observed. The optimum temperature for this study was found to be 37°C (Fig. 5b). According to previous studies, it is observed that the optimal temperature of L-glutaminase differs with the type of microorganism used^[33]. Addition of 1% carbon source into the production medium showed the significant increase in the enzyme level (L-glutaminase). Arabinose promoted maximal L-glutaminase yield. Other carbon source showed reasonable production of enzyme. Sucrose was observed to have the least yield of enzyme (Fig. 5c). Addition of 1% nitrogen source along with the production medium altered the production rate of L-glutaminase enzyme. Among all the nitrogen sources used, it was inferred that casein produced the highest amount of enzyme whereas ammonium nitrate showed least amount of enzyme activity (Fig. 5d). Salt-tolerant marine bacterial strain Pseudomonas aeruginosa CG-T8 showed highest L-glutaminase enzyme activity at optimal temperature 37°C and pH 5 which is similar to our results^[39]. Iyer and Singhal demonstrated that using Response Surface Methodology (RSM) for optimization resulted in a different optimized media and enzyme activity of 119 ± 0.12 U/mL^[40]. In another previous study, terrestrial fungal strain Fusarium oxysporum was used to produce L-glutaminase enzyme. The maximum enzyme production was 27.78 U/mL and optimum temperature was achieved at 35°C, pH 6 and 0.025% of glutamine concentration^[41].

CONCLUSION:

Marine yeasts are ubiquitous in nature with applications in various fields. From the current study, we can conclude that marine yeasts are potential producer of L-glutaminase enzyme which is known to have anti-cancer agents. A total of 26 marine yeast isolates were recovered from the collected samples. The isolates DAMB1, DAMB2, DAMB3 and DAMB4 showed positive result forming a pink zone in the media. Out of them, based on their potentiality to produce the L-glutaminase enzyme, yeast isolates DAMB1 and DAMB3 were further subjected to quantitative analysis through secondary screening. DAMB1 showed highest L-glutaminase activity. Optimal conditions for L-glutaminase production was seen at pH 8.0, 37°C with arabinose as carbon source and casein as nitrogen source. The potential marine yeast isolate was characterized microscopically using Gram’s staining and SEM analysis. 18s rRNA sequencing was done to identify the potential strain which was found to be Rhodotorula sp. DAMB1 (Acc. No. MH434671).

ACKNOWLEDGEMENT:

The Authors are very much thankful to the whole management and staff of VIT University, Vellore, Tamil Nadu, India for supporting this study and facilities including SEM (DST-FIST/VIT SEM) for completion of the study.

CONFLICT OF INTEREST:

All of the authors declare that there is no conflict of interest in this study.

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Received on 19.06.2019 Modified on 25.07.2019

Research J. Pharm. and Tech. 2020; 13(1): 209-215.

DOI: 10.5958/0974-360X.2020.00042.6