Process Optimization of L-Glutaminase Production; a Tumour Inhibitor from Marine Endophytic Isolate Aspergillus sp. ALAA-2000

Process Optimization of L-Glutaminase Production; a Tumour Inhibitor from Marine Endophytic Isolate Aspergillus sp. ALAA-2000 Mervat Morsy Abbas Ahmed1,2*, Taher M Taha3,4, Nageh F Abo-Dahab3 and Fareed SM Hassan3 1Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University (KAU), Saudi Arabia 2Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Giza, Egypt 3Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Assuit, Egypt 4Biology Department, Faculty of Science and Arts, Al Baha University, Saudi Arabia


Introduction
L-Glutaminase (L-glutamine amidohydrolase E.C 3.5.1.2) catalyses the hydrolysis of L-glutamine to glutamic acid and ammonia. In recent years, glutaminase has gained much attention due to their potential applications in pharmaceuticals as an anti-leukemic agent [1,2], flavor enhancing agent [3] and an efficient anti-retroviral agent [4]. Another most promising application of glutaminase is in biosensors for monitoring glutamine levels in mammalian and hybridoma cell cultures without the need of separate measurement of glutamic acid [5]. L-Glutaminase is widely distributed in animal tissues, plants and in a variety of microorganisms including bacteria, fungi and yeast [6][7][8] of which the most potent producers are fungi [9]. On an industrial scale, glutaminases are produced mainly by Aspergillus and Trichoderma [10][11][12][13]. The marine endophytic microflora in the coming decades will be the nature's best source of chemicals. Natural products metabolized from endophytic microorganisms represent desirable sources for effective therapeutic enzymes [8,14,15]. Marine fungi are rich profile of biologically active metabolites, especially from genera Penicillium, Aspergillus and Fusarium have been used aiming the development of novel therapies for treating cancer. Whereas the marine fungi are least studied than terrestrial counterparts and other ecological group, they have to study due to their production of new metabolites which are not found in terrestrial fungi [16].
Different methods of fermentation technology can be applied for the production of L-glutaminase. Commercial production of L-glutaminase had been carried out using submerged fermentation (SmF) technique [17,18]. But nowadays, solid state fermentation (SSF) has been emerged as a promising technology for the development of several bioprocesses and products including the production of therapeutic enzymes on a large-scale [19]. The primary advantage of SSF is the fact that many metabolites are produced at higher concentration.
Thus the present study, focuses on the L-glutaminase production as potent anticancer agents from a potential of culturable marine endophytic isolate Aspergillus sp. ALAA-2000 isolated from the internal healthy tissue of marine invertebrates as well as develop an economically viable bioprocess for production of L-glutaminase by evaluating and optimizing process parameters through manipulating the nutritional and physical parameters using low cost substrates.

Microorganism and culture maintenance conditions
Eighteen fungal isolates used in this study were isolated from Egyptian marine sponge Aplysina fistularis, a rich source of endophytic microorganisms on the isolation media peptone yeast extract glucose agar, potato dextrose agar and malt agar [20]. The fungal cultures were purified, maintained at 4°C until use and examined for the production of L-glutaminase enzyme.

Screening of fungal isolates for L-glutaminase productivity
Screening of fungal isolates for L-glutaminase activity was performed using the modified Czapek Dox medium (glucose 2 g, L-glutamine 10 g, KH 2 PO 4 1.52 g, KCl 0.52 g, MgSO 4 .7H 2 O 0.52 g, FeSO 4 .7H 2 O 0.01 g, agar 20 g, and distilled water 1000 mL) for the plate assay, 3 mL of 2.5% stock solution of phenol red in ethanol (pH 6.2) was added to 1000 mL of Czapek Dox medium. After 72 h of incubation at 26 ± 1°C, the appearance of a pink zone around the fungal colony in an otherwise yellow medium indicated L-glutaminase activity. The fungus which shows highest productivity was subsequently identified and selected to study the optimal conditions for L-glutaminase production in submerged and solid state fermentation.

L-Glutaminase assay
The activity of L-glutaminase is determined by estimating the amount of NH 3 liberated from glutamine. 0.5 ml of enzyme preparation was added to 0.5 ml of 0.04 M L-glutamine 0.5 ml distilled water and 0.5 ml of 0.1 M phosphate buffer (pH 8) then incubated at 37°C for 30 min. After incubation, 0.5 ml of 1.5 M trichloroacetic acid was added to stop the enzymatic reaction. Blank was run by adding the enzyme preparation after the addition of trichloroacetic acid. 0.1 ml of above mixture was taken and added to 3.7 ml of distilled water followed by addition of 0.2 ml Nessler's reagent. Absorbance was measured at 450 nm using a visible spectrophotometer. One international unit of L-glutaminase (U) was defined the amount of enzyme that liberates 1 μmol of ammonia from glutamate under optimum assay conditions. The enzyme yield was expressed as units/ml according to Imada et al. [17].

Protein estimation
Protein was determined by the absorbance at 280 nm using bovine serum albumin (BSA) as the standard [21]. All the sets have been performed in triplicates.

Effect of different natural wastes and leaching agents on L-glutaminase production
The production of L-glutaminase by Aspergillus sp. ALAA-2000 under solid state fermentation was estimated in 250 ml Erlenmeyer flasks containing 5 g of solid substrate; sugar cane bagasse, wheat bran, corn cobs, soy bean, kidney bean bran, wheat hay, rice bran, rice straw and corn casing separately moistened with mineral salt solution 10 ml of slates solution (glucose 0.6%, KH 2 PO 4 0.1%, MgSO 4 .7H 2 O 0.05 and KCl 0.05%) the flasks were autoclaved, cooled, inoculated with spore suspension and incubated under static condition for 6 days fermentation period each fermented substrate was extracted with different leaching agent [22]. The leaching out of L-glutaminase from the fermented solids was carried out with different extractants such as hot water (40°C), ethanol, acetone, Tween 80 (1%), sodium chloride (0.1%), citrate buffer pH 3, phosphate buffer pH 7, glycine buffer pH 12, and at a ratio of 1:5 (w/v).

L-Glutaminase activity under submerged fermentation
For the production of L-glutaminase by Aspergillus sp. ALAA-2000, different fermentation media as a modified Czapek Dox medium, mineral salts and starch nitrate ware applied. Aspergillus sp. ALAA-2000 strain was incubated in 250 ml Erlenmeyer flasks containing 50 ml of fermentation medium supplemented with L-glutamine and incubated at 27°C and 120 rpm on a rotary shaker for 4 days. At the end of fermentation period the clear supernatant after centrifugation at 4000 rpm for 20 min was used as enzyme preparations.

Optimization of the culture condition for L-glutaminase production under submerged fermentation
Various process parameters that enhance the yield of L-glutaminase by Aspergillus sp. ALAA-2000 strain in a modified Czapek Dox broth medium under shaking were investigated. The impact of incubation time (2-14 days), initial pH (3-9, adjusted with 1 M HCl or NaOH), and incubation temperatures (20-37°C) were evaluated. Moreover, the effect of incorporation of additional various carbon sources (10 g/L) separately in modified Czapek Dox broth medium (raffinose, xylose, mannitol, mannose, sucrose, maltose, sorbose, lactose, galactose, fructose, starch, pectin, cellulose and dextrose) instead of its carbon source (glucose). Organic nitrogen sources; beef extract, yeast extract, peptone and urea as well as inorganic nitrogen sources; ammonium nitrate, ammonium sulfate, ammonium hydrogen citrate were examined for their ability separately to stimulate the enzyme production in modified Czapek Dox broth medium instead of L-glutamine as N-base. Ten amino acids (lysine, isoleucine, glycine, thiamine, arginine, treptophan, proline, glutamic acid, cysteine and methionine) were examined as a sole nitrogen source for L-glutaminase production. Each of them was added to the medium instead of its nitrogen source in such amount that the final concentration of N-base remained unchanged. Different concentrations (0.025%, 0.05%, 0.1%, 0.15% and 0.2%) of the most suitable amino acid source were tested for their ability to enhance the L-glutaminase production for Aspergillus sp. ALAA-2000 strain. Some element supplementations (MgCl 2 , LiCl, CaCl 2, K 2 HPO 4 , BaCl 2 and NaCl) were supplementation to the modified Czapek Dox broth medium. Different concentrations (0.025%, 0.05%, 0.1%, 0.15% and 0.2%) of the most suitable element supplementation were tested for their ability to enhance the enzyme production by Aspergillus sp. ALAA-2000 strain. All the experiments were conducted in triplicate, subsequently and the mean values are considered.

Purification and characterization of L-glutaminase
The concentrated enzyme was subjected to ammonium sulphate fractionation with concentration ranging between 20% and 100% according to the method of Gomori [23]. The precipitate of crude enzyme was dissolved in a minimum volume of 0.2 M phosphate buffer (pH 6.0) and dialyzed overnight in a dialysis bag against the same buffer at 4°C. The L-glutaminase from ammonium sulphate precipitation (80%) was loaded onto the DEAE-cellulose column chromatography which that equilibrated and eluted with 0.02 M phosphate buffer (pH 8.0). The column was washed with four to five bed volumes of 0.02 M phosphate buffer (pH 8.0). The bound protein was containing linear gradients of NaCl (0.1, 0.25, 0.5, 1.0 M). The flow rate was 0.5 ml/min. The fractions eluted at each NaCl concentration were collected, pooled and tested for protein and L-glutaminase activity [24]. The molecular weight of the purified L-glutaminase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli [25].
The optimum reaction time for the partially purified enzyme was estimated at different times 10-100 min for L-glutaminase and then assayed by the direct nesslerization method. The optimum pH was determined by measuring enzymatic activity at 37°C using different buffers (0.1 M) with various pH values as: citrate-phosphate buffer (pH 3, 4, 5 and 6), phosphate buffer (pH 6-7), Tris-HCl buffer (pH 8-9) and glycine-NaOH buffer (pH [10][11][12]. Optimum temperature was assayed by measuring activity with varying temperatures (27,37,47,57,67,77 and 87°C) at the optimum pH values. enzyme at various temperatures (20-90°C) for 60 min and then the residual activity was determined at optimum pH and temperature. Partially purified L-glutaminase was incubated individually with different concentrations of L-glutamine in the reaction mixture (0.29, 0.73, 1.46, 2.92, 4.38 and 5.85 mg/ml) to find out the best substrate concentration for enzymatic assay under optimized assay conditions. Different concentrations (0.05, 0.125, 0.25, 0.5, 0.75 and 1.0 ml) of the partially purified L-glutaminase were incubated individually with the optimization concentration substrate solution in the reaction mixture to determine the best enzyme concentration under the optimized assay condition.
The effect of different metal ions including Na + , Ca 2+ , Mn 2+ , Mg 2+ , Co 2+ , Ba 2+ and Ni 2+ at a final concentration of 1.0 mM as well as EDTA at different concentrations (0.0001, 0.001 0.005, 0.01, 0.05 and 0.1 M) separately on purified L-glutaminase were studied. Residual activities in the presence of each chemical was assayed and compared with the control (without additions), which considered as 100% activity.

Sccrening and production of L-glutaminase
This study proved that Egyptian marine sponge Aplysina fistularis is a rich source of endophytic microorganisms. Whereas 18 fungal isolates were obtained on the isolation media peptone yeast extract glucose agar, potato dextrose agar and malt agar. All these marine endophytic isolates were evaluated as fruitful source of the therapeutic antitumor enzymes L-glutaminase. The activity of L-glutaminase enzyme was detected in all fungal strains. The fungal strain under the isolation number, ALAA-2000 was the most potent active producer. Therefore, this strain was identified and selected for furtherstudy. According to the analysis of ITS region sequence, together with its phenotypic and characteristics, the producing strain ALAA-2000 was identified as Aspergillus sp. and designated as Aspergillus sp. ALAA-2000 [20]. The screening and identification of filamentous fungi capable of secreting extracellular enzymes with biotechnological potential are activities of great importance [26]. Microbiology of sponges and corals can led to a huge number of other enzymes that applied in biotechnology such as cellulase, xylanase, keratinase, glucoamylase, pectinase, lipolytic activity, non-specific peroxidases, laccases, chitinase and glucanase [27][28][29][30][31][32]. L-glutaminase activity varied in fungal strains obtained through different studies. In the study of Sajitha et al. [33] from 50 different endophytic fungi isolated from green and red seaweeds, 10 isolates produced L-glutaminase activity. Among 400 marine isolates screened from various marine niches, one isolate showed productivity of L-glutaminase [7] and among twenty one isolates of Aspergillus wentii (38%) were capable of producing L-asparaginase and L-glutaminase [34]. The anticancer enzyme L-glutaminase are produced by a variety of fungi but the exclusive production of this enzyme in industry have been achieved mainly by Aspergillus species as A. fumigatus, Aspergillus sp. KUFS20, A. terreus MTCC 1782 [35][36][37].

Production of L-glutaminase; antitumor enzyme under different fermentation processes
When we used different natural sources as solid substrate fermentation such as sugar cane (bagasse), wheat bran, corn cobs, soy bean, kidney bean bran, wheat hay, rice bran, rice straw and corn casing, as are valuable and cheap energy sources of L-glutaminase productivity, with using different leaching agents such as hot water 40°C, ethanol, acetone, sodium chloride (0.1%), phosphate buffer (pH 7), glycine buffer (pH 12), Tween 80 (1%) and citrate buffer (pH 3) shown in Table 1. The hot water 40°C was the best leaching agent extracted of soy bean for L-glutaminase production (21.89 U/ml) followed by soy been extracted with phosphate buffer (20.14 U/ml), soy bean extracted with acetone (19.69 U/ml), wheat bran leached by NaCl (17.79 U/ml), soy bean leached with NaCl (17.65 U/ml), soy bean leached with citrate buffer (17.00 U/ml), soy been extracted with Tween 80 (16.92 U/ml), wheat bran extracted with citrate buffer (16.00 U/ml), and sugar can baggase extracted with NaCl (15.47 U/ml), respectively. Consequently agro industrial residues proved to be promising sources for the industrial production of this therapeutic enzyme, especially soybean, wheat bran and sugar can baggase. Fermentation technology has been widely used for the production of a wide variety of substances of industrial, medical and agriculture. Fermentation technique must be economic and environmental advantages. Kiruthika and Saraswathy [38] supported soy bean as solid substrate in SSF for L-glutaminase production by Vibrioazureus JK-79 whereas Nathiya et al. [35] reported the maximum L-glutaminase form A. fumigatus was produced with paddy straw; El-Sayed [10] by using different agro-industrial byproducts as solid substrates for induction of L-glutaminase by T. koningii found that wheat bran was the best substrate (12.1 U/mg protein). Furthermore, out of different agro industrial residues, A. flavus (FGNAS-7) produced the highest amount of L-glutaminase with ragi straw [39]. Moreover, Negi and Banerjee [40] tested various parameters such as leaching agent selection, amount of leaching agent, soaking time, and temperature in order to determine optimum extraction conditions of enzymes produced by A. awamari nakazawa MTCC 6652 and optimum conditions were achieved in a 10% glycerol. On the other hand, the effect of different three broth culture media named modified Czapek-Dox, mineral salts, and starch nitrate provided with 1% L-glutamine on L-glutaminase production by Aspergillus sp. ALAA-2000 were applied. Modified Dox medium showed the highest L-glutaminase activity (91.92 U/ml), while starch nitrate medium showed the lowest L-glutaminase activity (73.24 U/ml). The highest level of L-glutaminase activity observed after two days incubation period. 14 days incubation period resulted in reduction of L-glutaminase to 0.73, 11.67 and 0.44 U/ml, respectively. These data clearly indicated that the L-glutaminase under study is extracellular enzymes. Extracellular enzymes are of potent important in industrial application in the terms of minimize the cost of production process.
The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients. An additional advantage of this technique is that purification of products is easier [41]. Elshafei et al. [42] and Siddalingeshwaraet al. [43] reported the production of L-glutaminase by P. brevicompactum NRC 829 and A. wentii KGSD4 mu under submerged fermentation, but Prasanna and Raju, [44] and Nathiya et al. [45] recorded solid state fermentation as the best fermentation technique for the production of L-glutaminase by A. oryzae NCIM1212 and A. flavus, respectively. The maximum L-glutaminase production by A. oryzae NCIM 1212 was recorded after 2 days of incubation period [44]. Conversely the highest L-glutaminase production by A. flavus (FGNAS-7) was obtained after 6 days [39].

Process optimization of L-glutaminase production under submerged fermentation
Effect of different pH and temperature values on L-glutaminase production from Aspergillus sp. ALAA-2000: To determine the optimum temperature and pH, this supported the highest L-glutaminase production by Aspergillus sp. ALAA-2000. The results ( Table 3) showed that pH 4 at 27°C supported the highest L-glutaminase production (219.14 U/ml). L-Glutaminase was gradually decreased at higher pH to reach 80.25 U/ml at pH 9 and 27°C. Whereas incubation of Beauria sp. and A. fumigatus at 27°C and 30°C supported maximum L-glutaminase production [5,35], initial pH of 4 and 6 ensured L-glutaminase biosynthesis in Streptomyces variabilis ASU319 and A. fumigatus [35,46], respectively but Prasanna and Raju [44] reported that among a wide range of either incubation temperatures and initial pH the maximal yield of L-glutaminase production by A. oryzae NCIM1212 was attained at 30°C and pH 7.
Effect of different concentrations of L-glutamine on L-glutaminase production: L-glutamine is the inducer for L-glutaminase production. Different inducer concentrations (0.5, 1, 1.5, 2 and 2.5%) were added to the modified Dox medium individually to determine the optimum concentration for the enzyme production (Table 4). L-glutamine at concentration of 2.5% supported the highest induction of L-glutaminase up 78.79 U/ml. The high yield of L-glutaminase (32.7 U/ml) produced by Streptomyces griseus when used L-glutamine as substrate due to L-glutamine is the specific inducers for L-glutaminase [47].

Effect of different nitrogen and carbon sources on L-glutaminase production:
The effect of different organic nitrogen sources such as peptone, beef extract, yeast extract, urea, and glutamine as well as inorganic sources such as ammonium sulphate, diammonium citrate, sodium nitrate, and ammonium nitrate were tested. Each one of these nitrogen sources was added to the modified Dox medium individually at concentration in comparison to the enzymatic inducer L-glutamine was evaluated. Results (      Table 4: Effect of different concentrations of substrate, nitrogen and carbon sources, supplementation elements as well as amino acids on L-glutaminase production. Aspergillus sp. ALAA-2000 strain was required the inducer L-glutamine for the best biosyntheses of L-glutaminase (109.43 U/ml). However beef extract and sodium nitrate showed lowest production of L-glutaminase (31.66 and 7.59 U/ml, respectively) by Aspergillus sp. ALAA-2000. Katikala et al. [48] reported that marine bacterial isolate LG24 gave the highest yield of extracellular L-glutaminase 22.68 U/ml in 120 h when L-glutamine supplemented as sole carbon and nitrogen source in the media.
On the other hand, to determine the best carbon sources for therapeutic enzyme production L-glutaminase produced by Aspergillus sp. ALAA-2000 grown in modified Dox medium supplemented with different carbon sources are represented in Table 4. It was obvious that dextrose increased the L-glutaminase production by Aspergillus sp. ALAA-2000 to 124.02 U/ml followed by mannose, sucrose, sorbose, pectin, galactose, cellulose, starch, mannitol, lactose, xylose, maltose and raffinose (121.09, 119. 20 Overall, all carbon sources did not exhibited much significant increase in L-glutaminase production due to Dox modify medium free carbon relatively yielded a high amount of L-glutaminase (119.64 U/ml). On the contrary, the yield of L-glutaminase from S. griseus was increased to 26.3 U/ml by utilized the galactose as the carbon source [47]. Table 4 showed effect lysine, isoleucine, glycine, thiamine, arginine, treptophan, proline, glutamic acid, cysteine, andmethionine on L-glutaminase production. All amino acids had stimulation effect on L-glutaminase production with different ratio ranged between 57.8% with cysteine to 36% with glutamic acid and then the impact of different concentrations of cysteine, the best amino acids for L-glutaminase production by the marine endophytic strain Aspergillus sp. ALAA-2000 was studied. Out of this concentration cysteine at a concentration of 0.1% supported the highest L-glutaminase production 135.98 U/ml. Amino acids were served as source of energy and carbon in addition to nitrogen as previously reported for L-glutaminase production by Fusarium sp. [46].

Effect of different amino acids on L-glutaminase production: Data illustrated in
Effect of addition and elimination of different supplementation elements on L-glutaminase production: Magnesium chloride enhanced L-glutaminase production by Aspergillus sp. ALAA-2000 to 97.75 % but LiCl, CaCl 2 , K 2 HPO 4 , BaCl 2 and NaCl decrease it to 74. 41, 71.49, 57.92, 77.62 and 78.79 U/ml, respectively (Table 4). Therefore, the effect different concentrations of MgCl 2 on L-glutaminase production were studied. L-glutaminase activity increased with the increasing of MgCl 2 concentration till 0.1% after that decreased. The main components of the modified Dox medium are KCl, MgSO 4 .7H 2 O, and KH 2 PO 4 . Elimination of these components from culture medium did not strongly inhibited L-glutaminase production. Inhibition ratio reached up to 6%, 3% and 22% with the elimination of KCl, MgSO 4 .7H 2 O, and KH 2 PO 4 , respectively. Na + enhanced of L-glutaminase production by A. fumigatus and A. oryzae NCIM 1212 [35,44].

Partially purification of L-glutaminase with DEAE-cellulose
The purification steps from the crude extract of Aspergillus sp. ALAA-2000 are summarized in Table 5 and Figure 1. L-Glutaminase was purified from the culture filtrate by 80% saturation of ammonium sulfate (the fraction showed the highest glutaminase activity), resulted in specific activity of 9.52 U/mg protein, 8.89 purification folds followed by DEAE-cellulose column chromatography. The purification of the glutaminase was increased 36.72 fold with overall yields of 37.42 %. The partially purified of L-glutaminase produced by P. brevicompactum NRC 829 has total activity 321.6 U, total protein 0.37 mg, Specific activity 869.08 (U/mg), purification 162.75-fold and yield 48.21% after Sephadex G-200 [42]. While Ali and Mohamed [49] reported that the total activity 44 U, total protein 0.33 mg, specific activity 133 U/mg, purification 230-fold and yield 25% of L-glutaminase produced by P. politans NRC510.

Characterization of partially purified L-glutaminase activity
Due to the purification of L-glutaminase showed the maximum single peak of L-glutaminase relative activity (107.2%) produced by Aspergillus sp. ALAA-2000 at 47°C refer to the mono meric nature of L-glutaminase enzymes. The thermo stability of L-glutaminase activities showed a wide range of L-glutaminase thermo stability from (30°C to 60°C) and higher than this temperature stability gradually decreased. Maximum thermo stability of L-glutaminase activity at 40°C and 50°C (100 and 100%), respectively and sharply decreases the activity (41%) at 90°C ( Table 6). The maximum activity of purified L-glutaminase from P. brevicompactum NRC 829 and S. variabilis were insured at incubation temperatures 30°C and 50°C, respectively [42,46]. Furthermore, Elshafei et al. [42] reported that L-glutaminase was stable at 50ºC to 60ºC for 60 min and it retained about 92% and 66% of its initial activity after incubation at 70ºC for 30 min and 80ºC for 5 min, respectively without the substrate.
The L-glutaminase produced by marine endophytic Aspergillus sp. ALAA-2000 strain exhibited classical pattern of pH activity relationship with pH optimum at pH10 (Table 6). Elshafei et al. [42] and Dura et al. [24] reported that the pH 7 and 8.5 were increased L-glutaminase activity purified from P. brevicompactum NRC 829 and Debaryomyces sp. CECT 11815, respectively. Figure 2 showed that, the activity of partially purified L-glutaminase was tested at 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 min incubation times. The results showed on peak of activity after 30 min of incubation and it was 100%. The best incubation time for L-glutaminase production by P. brevicompactum NRC 829 and S.variabilis was 60 min [42,46].
The effect of different concentrations of L-glutamine 0.29, 0.73, 1.46, 2.92, 4.38 and 5.85 mg/ml on the activity of L-glutaminase were detected. The activity of L-glutaminase was increased with increasing substrate concentration ratio to 4.38 mg/ml of L-glutamine (114.1 U/ml). Increasing substrate concentration resulted decreasing in the activity of L-glutaminase. Different partially purified enzymes concentrations, 0.05, 0.125, 0.25, 0.5, 0.75 and 1 ml of L-glutaminase were attempted in the enzymatic reaction with glutamine at concentration of 0.75 mg/ ml. The suitable enzyme concentration in the reaction mixture that supported the highest activity was 113.4% for L-glutaminase produced by 1ml. Elshafei et al. [42] and Ali and Mohamed [49] reported the high affinity of L-glutaminase activity produced by P. brevicompactum NRC 829 and P. politans NRC510 with 1.66 mM and 10 μmol concentrations of L-glutamine, respectively.
The effect of Ni 2+ , Ba 2+ , Ca 2+ , Na + , Co 2+ , Mn 2+ and Mg 2+ with final concentration of 0.5 mM of each cation on partially purified L-glutaminase activities was studied. Compared to control Ba 2+ , Ca 2+ , Na + , Co 2+ , Mn 2+ and Mg 2+ increase the L-glutaminase activity by 15.7, 6.8, 41.7, 15.4, 9.4 and 15.8, respectively but Ni 2+ achieved decrease enzyme activity. The maximum activator cation is Na + by 41.7% compared with control. On the other hand, the effect of different EDTA concentrations (0.0001, 0.001, 0.005, 0.01, 0.05 and 0.1 M) on the activity of partially purified L-glutaminase was studied. Results indicated that different EDTA concentrations have no effect on L-glutaminase activity which means that L-glutaminase enzymes was represent as a non metallicenzyme. Ali and Mohamed [49] and Elshafei et al. [42] reported that sodium chloride was the best activator for L-glutaminase activity from P. politans NRC 510 and P. brevicompactum NRC 829. Also, Elshafei et al. [42] reported L-glutaminase produced by P. brevicompactum NRC 829 as non metalloenzyme but Abd-Alla et al. [46] reported L-glutaminase produced by S.variabilis as metallo enzyme.

Conclusion
The potential of isolated marine endophytic strain Aspergillus sp. ALAA-2000 for L-glutaminase production was analyzed under different fermentation modes with different process parameters. Maximum production was supported with L-glutamine, dextrose,