Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 18  |  Issue : 1  |  Page : 27-41

Biochemical studies and biological activities on L-glutaminase from rhizosphere soil Streptomyces rochei SAH2_CWMSG


1 Chemistry of Natural and Microbial Products, Department Pharmaceutical and Drug Industries Research Division, National Research Centre, Giza, Egypt
2 Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre, Dokki, Giza, Egypt

Date of Submission02-Jul-2018
Date of Acceptance29-Aug-2018
Date of Web Publication26-Mar-2019

Correspondence Address:
Hassan M Awad
Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, PO Box: 12622, Dokki, Giza
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/epj.epj_32_18

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  Abstract 

Background and objective L-glutaminase (L-GLUNase) is a potential anticancer enzyme that hydrolyzes amide bond of L-glutamine to give glutamate and ammonium ion. It is used as an antioxidant, a flavor enhancing agent and a biosensor for glutamine level measurement. The aim was to produce L-GLUNase in high yield from a promising local Streptomyces isolate for many pharmaceutical applications.
Materials and methods A total of 20 Streptomyces isolates for their capacity of L-GLUNase production were screened. A potent L-GLUNase producer, SAH2_CWMSG isolate, was identified by phenotypic and phylogenetic analysis. L-GLUNase was purified using ammonium sulfate followed by gel filtration on Sephadex G-100. The purified L-GLUNase was characterized, and its application as an antimicrobial, anticancer, and antioxidant agent was investigated.
Results and conclusion The phylogenetic analysis of SAH2_CWMSG strain confirmed that the SAH2_CWMSG strain was most similar to Streptomyces rochei (99%). It produced L-GLUNase activity of 58 U/ml under shake flask submerged fermentation. The purified L-GLUNase has the molecular weight of 55 kDa and Km and Vmax value of 1.314 mmol/l and 95.24 μ Me/min, respectively. Of the various physiochemical parameters tested, pH 7.5 and temperature 40°C were optimal for the enzyme activity. On the contrary, 10 mmol/l of Mn+2 showed a slight increase in L-GLUNase activity. A promising Streptomyces sp. fully identified as S. rochei SAH2_CWMSG (Gen Bank ID: KU720627) is an efficient source of L-GLUNase production. Therefore, it can be potentially used as enzyme supplement, which has many industrial and pharmaceutical applications.

Keywords: biological activities, L-glutaminse characterization, phenotypic and phylogenetic identification, Streptomyces rochei SAH2_CWMSG


How to cite this article:
Awad HM, El-Deen AM, Mostafa ESE, Hassabo AA. Biochemical studies and biological activities on L-glutaminase from rhizosphere soil Streptomyces rochei SAH2_CWMSG. Egypt Pharmaceut J 2019;18:27-41

How to cite this URL:
Awad HM, El-Deen AM, Mostafa ESE, Hassabo AA. Biochemical studies and biological activities on L-glutaminase from rhizosphere soil Streptomyces rochei SAH2_CWMSG. Egypt Pharmaceut J [serial online] 2019 [cited 2019 Jul 17];18:27-41. Available from: http://www.epj.eg.net/text.asp?2019/18/1/27/254964


  Introduction Top


L-glutaminase (L-glutamine amidohydolase E.C 3.5.1.2) is a potential anticancer enzyme that hydrolyzes amide bond of L-glutamine to give glutamate and ammonium ion. L-glutaminase assumes a noteworthy part in the nitrogen metabolism of both prokaryotes and eukaryotes. L-GLUNase has pulled in much attention as of late for its wide application in pharmaceuticals as well as being a hostile agent toward leukemia [1]. L-GLUNase exhibits its anticancer effect by depleting L-glutamine from the tumor cells, prompting their death as they are dependent on this amino acid [2]. For example, L-GLUNase purified from Purified L-GLUNase obtained from Aspergillus flavus could stop a breast carcinoma [3].

L-GLUNase is actually a vital ordinary antioxidant that helps in avoiding human infections. Moreover, it is not related to lethal and cancer-causing effects like those of artificial antioxidants [4],[5], not very many reports are accessible for the utilization of bacterial and fungal L-GLUNases as antioxidant mediators [4],[5],[6].

L-GLUNase is also used as an efficient antiretroviral agent [7], along with its use in food industry as a flavor and aroma-enhancing agent [8]. Another important application of glutaminase is that it also plays an important role in biosensor as a monitoring agent for glutamine level measurement [9].

It can be obtained from, animal, plant and microbial cells. Nevertheless, the greatest sources of it are the microorganisms, demonstrating the simple generation of a coveted compound in significant sum with soundness and hereditary material control [10]. Among the microorganisms producing bioactive compounds, 73% are Streptomyces and 27% being rare actinomycetes. Streptomyces are well recognized to produce valuable medicines, particularly antibiotics and anticancer mediator, and industrial products like enzymes for revenue-generating discovery platform [11],[12]. A lot of information has been presented for microbial L-GLUNase producers like Streptomyces rimosus [13], Streptomyces avermitilis and Streptomyces labedae [14], Streptomyces gresius [15], Streptomyces enissocaesilis DMQ-24 [4], and Streptomyces canaries [16].

In the present work, an endeavor was made to isolate and fully identify an efficient local Streptomyces isolate for L-GLUNase production in high yield, representing a promising isolate. The purified enzyme was characterized and its biological activities were investigated.


  Materials and methods Top


Isolation, screening and quantitative assay of L-glutaminase

Five soil samples were collected in sterile bags from the rhizosphere area of wheat plants at Mashtool El Sook, El Sharkia Governorate, Egypt. Overall, 100 μl of each serial dilution up to 10-7 was taken and spread on starch nitrate agar (SNA) plates and incubated at 28°C for 5–7 days [17]. After incubation, the pure individual Streptomyces colonies were picked out, maintained in ISP-2 agar slant and kept at 4°C.

Screening of isolates for L-GLUNase activities in minimal glutamine agar medium containing of 0.009% w/v of phenol red was done. Isolates showing maximum zone of color change (from yellow to pink) were selected as a possible strain for further studies [18].

Potential Streptomyces isolate identification

Conventional taxonomy

Morphological, biochemical, cultural, and physiological characterization of the potential strain SAH2_CWMSG was performed and recommended by the International Streptomyces Project (ISP) [19]. Microscopic depiction was performed with cover slip culture. Formation of aerial, substrate mycelium and spore arrangements on mycelium was monitored under a phase-contrast microscope (Nikon Eclipse E600, New York, USA) at 200 magnification and scanning electron microscopy (JEOL JSM 5300; JEOL Techniques Ltd, Tokyo, Japan). The cultural characteristics such as growth, coloration of aerial and substrate mycelia and formation of soluble pigment were investigated in seven different media, including ISP-2–ISP-7 using the procedures as recommended by the ISP. Biochemical and physiological characterization analyses such as melanin pigment production, nitrate reduction, gelatin liquefaction, and starch hydrolysis were also performed as suggested by the ISP. The growth rate in NaCl (2–13%) and survival at 50°C were also evaluated. Furthermore, the capability of the isolates to utilize various carbon and nitrogen sources was estimated using ISP-9 agar medium. Based on the aforementioned characteristics, genus level identification of the potential strain was made by Bergey’s Manual of Systematic Bacteriology [20].

Disk diffusion susceptibility test

The disk diffusion susceptibility of the selected isolate was carried out according to Jorgensen and Turnidge [21]. The test was performed by applying SAH2-CWMSG strain inoculum to a SNA plate. Six standard antibiotic disks, for example, tetracycline, neomycin, oxytetracycline, vancomycin, rifamycin, and streptomycin (Bioanalyse, Ankara, Turkey), were placed on the inoculated agar surface. Plates were incubated for 24–48 h at 28°C before measurement of results. The zones of growth inhibition around each of the antibiotic disks were measured to the nearest millimeter.

Phylogenetic identification

DNA isolation and PCR amplification

An overnight culture of the SAH2-CWMSG strain grown at 28°C was used for the preparation of genomic DNA. DNA extraction was done by using the protocol of the Gene JET genomic DNA purification Kit (Thermo K0721; Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) following the manufacturer’s instructions on the kit. The PCR amplification of the 16S rDNA region was carried out following the manufacturer’s instruction regarding the Maxima Hot Start PCR Master Mix (Thermo K1051). The 16 s rDNA was amplified by PCR (PCR system 9700; Applied Biosystemes, Perkin–Elmer, Foster City, California, USA) using primers designed to amplify a 1500 bp fragment of the 16S rDNA region. The domain bacteria-specific primer 27 F (forward primer) was 5′-AGAGTTTGATCMTGGCTCAG-3′ and the universal bacterial primer 1492 R (reverse primer) was 5′-TACGGYTACCTTGTTACGACTT-3′ [22].

The PCR reaction was performed with 5 µl of genomic DNA as the template, 1 µl of 16 s rRNA forward primer, 1 µl of 16 s rRNA reverse primer, 18 µl water, nuclease-free and 25 µl Maxima Hot Start PCR Master Mix (2×) in a 50-µl reaction mixture as follows: activation of 2 Taq polymerase at 95°C for 2 min, 35 cycles of 95°C for 1 min, and 65°C and 72°C for 1 min each, and finally, a 10-min steep at 72°C. After completion, the PCR products were electrophoresed on 1% agarose gels, containing ethidium bromide (10 mg ml/l), to ensure that a fragment of the correct size had been amplified.

DNA sequencing, phylogenetic analysis and tree construction

The 16S rRNA sequence analysis and phylogenetic tree construction by neighbor-joining method was performed for species-level confirmation of Streptomyces spp. The classifier was trained on the new phylogenetically consistent higher-order bacterial taxonomy (Ribosomal Database Project, RDP Classifier) proposed by Wang et al. [23], (http://rdp.cme.msu.edu/classifier/classifier.jsp).

The amplification products were purified with the K0701 Gene JET PCR Purification Kit Thermo (Thermo Fisher Scientific Inc.). Afterwards, the samples become ready for sequencing in an ABI Prism 3730XL DNA sequencer and analyzer of GATC Company (GATC Biotech Ltd., London, UK). Sequencing reaction was performed with the primers 518 F 5′-CCA GCA GCC GCG GTA ATA CG-3′ and 800 R 5′-TAC CAG GGT ATC TAA TCC-3′ using a PRISM Big Dye Terminator v3. 1 Cycle sequencing Kit. The DNA samples containing the extension products were added to Hi-Diformamide. The mixture was incubated at 95°C for 5 min, followed by 5 min on ice and then analyzed by ABI Prism 3730XL DNA analyzer (Applied Biosystems). The sequence alignment was prepared with DNASTAR software programs (DNASTAR Inc., Madison, Wisconsin, USA).

Phylogenetic data were obtained by aligning the nucleotides of different 16S RNA retrieved from the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST), using the CLUSTAL W program version 1.8 with standard parameters. The sequences with 98–100% homology were considered for molecular taxonomy analysis. Multiple sequence alignment was performed in the 16S rRNA sequence generated in this study and sequences from GenBank database with the CLUSTAL W program. Phylogenetic tree was constructed using the neighbor-joining and maximum-parsimony tree making methods in Mecular Evolutionary Genetic Analysis (MEGA version 6.0) software [24], based on bootstrap values of 500 replications.

Production medium and cultivation conditions for L-glutaminase

The broth medium was used in this study for primary evaluation of L-GLUNase activity (g/l): glucose, 3; NaH2PO4, 6; K2HPO4, 3; NaCl, 0.5; MgSO4, 0.5; CaCl2, 0.015; and glutamine, 3. The pH of the medium was adjusted to 7.0 before sterilization. The carbon source and amino acid were sterilized separately and added to the fermentation medium before inoculation. Overall, 50 ml of the liquid medium was dispensed into 250-ml Erlenmeyer flask and autoclaved at 121°C for 20 min. The flasks were inoculated in duplicates with 5% of the vegetative cells from a 7-day-old culture. The inoculated flasks were incubated at 28°C on a rotary shaker (New Brunswick Scientific Co., Edison, New Jersey, USA) at 200 rpm for 120 h. The uninoculated fermentation medium was used as a negative control during the experiment. The contents of each flask were harvested by centrifugation at 10.000 rpm for 10 min, and the supernatant was analyzed for enzyme activity and cell growth.

Assay of L-glutaminase

L-GLUNase activity was determined using the method of Imada et al. [25], utilizing L-glutamine as substrate, and the released ammonia was measured using Nessler reagent. An aliquot of 0.5 ml of the sample was mixed with 0.5 ml of 0.04 M L-glutamine solution in the presence of 0.5 ml of distilled water and 0.5 ml Tris-HCl buffer (0.05 mol/l; pH 8). Then the mixture was incubated at 37°C for 30 min, and the reaction was stopped by the addition of 0.5 ml of 1.5 mol/l Tri-chloroacetic acid. To 0.1 ml of the mixture, 3.7 ml of distilled water and 0.2 ml of Nessler’s reagent were added and incubated for 10 min at room temperature. A standard graph was plotted using ammonium sulphate. One international unit of L-GLUNase was defined as the amount of enzyme that liberates 1 µmol of ammonia per milliliter per minute.

The protein content measurement

The protein content was estimated by using the Lowry et al. [26] with BSA (1 mg/ml) as standard.

Extraction and purification of L-glutaminase

The bulk-produced culture broth under optimal conditions was chilled and centrifuged at 10 000 rpm for 10 min to remove the cell debris and the supernatant, referred as cell-free extract. The enzyme was precipitated by slow addition of ammonium sulphate (25–100%) by continuous gentle stirring on a magnetic stirrer overnight at 4°C with occasional changes of Tris buffer pH 8. The chilled mixture was then subjected to centrifugation at 15 000 rpm for 15 min. The supernatant was discarded carefully, and the precipitate was dissolved in a minimum amount of 0.05 mol Tris-HCl buffer pH 8.0. The precipitated protein obtained after treatment of the crude enzyme extract with ammonium sulfate (75% saturation) was dissolved in 0.05 mol Tris-HCl buffer pH 8 and dialyzed (with gentle stirring) in cold same buffer for 24 h with changing the buffer four times. After dialysis, the enzyme solution (1 ml containing 13.3 mg protein and with a total activity of 405.6 units) was applied to Sephadex G-100 column, and the protein was eluted by the identical buffer at a flow rate of 60 ml/h [27].

Physiochemical characterization of purified L-glutaminase

Effect of temperature on enzyme activity and stability

The effect of reaction temperature on L-GLUNase activity was tested by incubating the reaction mixture at different temperatures ranging from 30 to 55°C in 0.05 mol Tris-HCl buffer pH 8. The thermal stability of the purified enzyme was determined by incubating the enzyme solution at different times (15–60 min) at various temperatures (40, 50, 60, 70, and 80°C) in the absence of substrate. The enzyme was removed and cooled to the room temperature, and the residual activity was measured by the standard assay method as previously mentioned.

Effect of pH on L-glutaminase activity and stability

The optimum pH for L-GLUNase activity was determined using 0.05 mol sodium citrate (pH 4.0–7.0), 0.05 mol potassium phosphate (pH 6.5–8.0) buffers and 0.05 mol Tris-HCL buffer (pH 7–9). After incubating each reaction at 45°C for 30 min, enzymatic activity was detected. The pH stability of the enzyme was determined by preincubating the enzyme solution at different pH values ranging from 4.0 to 9.0 pH with 0.05 mol sodium citrate buffer for 2 h at 4°C. At the end of the preincubation time, the pH value of enzyme solution was readjusted to pH 7.5 and then residual enzyme activity was assayed by the standard method.

Reaction time for enzyme activity

L-GLUNase was incubated with its substrate; the greater amount of product will be formed. The reaction of the enzyme was preceded from 0 to 30 min. The enzyme activity was assayed by the standard method to select an appropriate incubation time.

Determination of purified L-GLUNase molecular weight by SDS-page

The purity of L-glutamine, protein was tested by SDS-PAGE using Coomassie brilliant blue dye using standard molecular markers according to the procedure of Laemmli [28] and Ali et al. [29].

Calculation of Km and Vmax values for L-GLUNase

To characterize the L-GLUNase produced by SAH2_CWMSG strain, the pure enzyme (10–100 mg/ml) was incubated for 30 min with different concentrations of glutamine (1–10 mmol/l). The Km and Vmax values of L-GLUNase were calculated from the graph of substrate concentration versus reaction velocity.

Effect of metal ions and other reagents on L-GLUNase activity

The effects of some metal ions, for example, magnesium chloride (MgCl2), potassium chloride (KCl2), magnesium chloride (MnCl2), ferrous chloride (FeCl2), cobalt chloride (CoCl2), sodium chloride (NaCl), nickel chloride (NiCl2); iodoacetate, SDS; glutathione; and EDTA on L-GLUNase activity were tested. The effect of these compounds on enzyme activity was assessed by incubating enzyme solution with 10 mmol/l concentrations of each compound for 20 min before addition of substrate. After a preincubation time, enzymatic activity was determined under optimal assay conditions.

Biological activities of L-GLUNase

Antimicrobial bioassay − well diffusion method

Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538),  Escherichia More Details coli (ATCC 7839), and Pseudomonas aeruginosa (ATCC 9027) were used as bacterial test strains. The bacteria were slanted on nutrient agar (Merck, Darmstadt, Germany). The antibacterial screening bioassay was made by the agar well diffusion method described by Jorgensen and Turnidge [21] using Mueller-Hinton agar (Lab M Limited, Bury, Lancashire, UK). On the contrary, Candida albicans (ATCC 10231), Saccharomyces cervecia (ATCC 9763), Aspergillus niger (ATCC 16404), Fusarium oxyspoium (ATCC 62506), Fusarium solani (ATCC 36031), and Alternaria spp. (ATCC 20084) were used as antifungal test strains. The fungal test strains were slanted and maintained on the potato Dextrose Agar medium (Lab M Limited).

Anticancer activity

Cell lines and culturing

Three human cancer cell lines are used throughout this study, namely, Caucasian breast adenocarcinoma (MCF-7), hepatocellular carcinoma (Hep-G2), and colon carcinoma (HCT-116) beside one normal cell line, namely, human epithelial retina cells, which were obtained from the American Type Culture Collection (Rockville, Maryland, USA). The tumor cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM for cancer cell lines) and DMEM-F12 for normal cell line, supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere containing 5% CO2. Cells were seeded at a concentration of 2×106 in a 25 ml tissue culture flask and incubated at 37°C till 80–90 confluent sheet.

In vitro ant proliferative assay

The anticancer activity was measured in vitro using the MTT assay according to the previously reported standard procedure [30],[31]. Cells were inoculated in 96-well micro-titer plate (104 cells/well) for 24 h before treatment with the tested compound to allow attachment of the cell to the wall of the plate. The tested pure enzyme was dissolved in DMSO at 1 mg/ml immediately before use and diluted to an appropriate volume just before addition to the cell culture. Cells were incubated alone or with enzyme at different concentration (1000, 500, 250, 125, and 62.25 μg/ml). After 48 h of incubation cell with enzyme, the cells were fixed, washed and stained for 30 min with 0.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and the attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. The results were compared with the antiproliferative effects of the reference control doxorubicin.

Antioxidant activity of the purified L-glutaminase by DPPH assay

The purified enzyme was tested for the scavenging effect on the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical according to the method of Sajitha et al. [6] with slight modifications. One milliliter of serial dilution (100–500 mg) of L-GLUNase was added to 8 ml of 0.004% (w/v) DPPH in ethanol (95%). The mixture was then incubated at 37°C for 60 min. The scavenging activity on the DPPH radical was determined by measuring the absorbance at 517 nm, using Agilent Technologies, Cary Series UV–Vis spectrophotometer (Agilent Technologies, Santa Clara, California, USA). Ascorbic acid was used as standard. The DPPH radical scavenging activity (% inhibition) was calculated by the following formula:




  Results and discussion Top


Isolation and detection of Streptomyces producing L-GLUNase

Approximately 20 isolates of Streptomyces were isolated from rhizosphere soils of wheat plant on SNA medium and tested on a medium containing glutamine and phenol red for L-GLUNase detection. Isolates growth and L-GLUNase production (pink zone) were observed.

Seven Streptomyces segregates developed well-shaped pink color around their growth. Of these seven isolates, SAH2_CWMSG was selected showing a dark pink color as presented in [Figure 1]. SAH2_CWMSG was isolated from the rhizosphere of the wheat plant, grown in Al Sharkia Governorate, Egypt.
Figure 1 Qualitative screening for L-GLUNase production for Streptomyces rochei SAH2_CWMSG.

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Identification of Streptomyces isolate

Conventional taxonomy of SAH2-CWMSG isolate

Morphological properties: [Table 1] demonstrates that the growth of the SAH2_CWMSG strain differs from weak to abundant based on the medium contents. The growth was feeble on media ISP-1, ISP-6, and nutrient agar and strong on the other media. The color of elevated mycelium extended from bright gray to dim gray. Thus, the aerial mycelium is appointed to the gray series. The substrate mycelium varied upon the medium constituent. The color of substrate mycelium was beige with ISP medium no. 1 and 6 and nutrient agar, and it was dark when utilizing ISP medium no. 2, 5, and 7. Spore masses were coordinated against the seven color wheels of Tresner and Backus [32], as utilized as a part of the ISP.
Table 1 Cultural characteristics of the Streptomyces rochei SAH2_CWMSG at 14 and 21 days

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Microscopically, it was noted that the morphology of the spore chains of aerial mycelium is of the spiral type ([Figure 2]a). As indicated by the state of the shape of the spore chains detected under light microscopy, the isolates were assembled as RF, spiral (S) and retinaculum a pertum (RA) [20]. The micrograph in [Figure 2]b demonstrates that the individual spores are cylindrical with a smooth surface, which was resolved by the classes of Tresner and Davies [33], who found that spore surface is one of the expressive portrayals for each kind of culture.
Figure 2 Light microscopy images of the aerial mycelium showing a rectus-flexible (straight spore chains) type (G×400) for 14 days at 28°C (a). Scanning electron micrographs showing smooth spore surface ornamentation (×7, 500) of Streptomyces rochei SAH2_CWMSG grown on starch nitrate agar medium for 21 days at 28°C (b).

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Physiological and biochemical characteristics

[Table 2] demonstrates that SAH2_CWMSG does not deliver melanin color, while it degraded protein and starch on the media utilized. Additionally, the results were positive for nitrate reduction, milk coagulation and gelatin liquefaction. Different classifications were tried and considered to build up the species classification of a new isolate as recognized by Holt et al. [34].
Table 2 Physiological, morphological, biochemical properties and amino acids utilization of Streptomyces rochei SAH2_CWMSG

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The cell wall of the isolate was affirmed to contain the ll-diaminopimelic acid (ll-DAP) type [35], a classic constituent of Streptomyces. Besides, the isolate can use all carbon sources of the examined, sugar utilizing ISP-9 by various degrees of the usage as represented in [Table 2]. Strain SAH2_CWMSG was growing well in a medium containing most of the amino acids used ([Table 2]). Moreover, a frail growth was noticed on medium including L-cysteine as a nitrogen source.

The outcomes in [Table 2] demonstrated that the abundant growth of SAH2_CWMSG was seen in a temperature scope of 26–45°C and the presence of 0–7% NaCl, whereas there was no growth at 50°C and 10–13% NaCl.

The characteristics of SAH2_CWMSG strain were matching with Streptomyces spp. published in Bergey’s manual with respect to morphological, physiological, and biochemical characters [19]. In brief, SAH2_CWMSG fit into the gray series group, with negative melanin pigment, spiral hyphae and a smooth spore surface. Classification and identification of Streptomyces established on morphological and biochemical characterization are insufficient in most cases; consequently, molecular information, mainly rRNA gene sequences, has been announced [34].

Genotypic identification of SAH2_CWMSG isolate

The 16S rRNA is a strong tool for phylogenetic analysis and species diversity of the genus Streptomyces. It can be used as a genomic technique in parallel to traditional taxonomic methods, including numerical, phonetic, and other genomic analyses [36]. Consequently, in this study, molecular biology methods depending on the 16S rRNA gene sequence of the isolate were partly sequenced in equivalent to traditional methods.

PCR amplification and phylogenetic analysis

The 16S rRNA gene of SAH2_CWMSG was amplified by the Streptomyces-specific PCR primers stated previously in materials and methods. The specificity of the PCR was influenced by components as the primers, the characters of the gene regions flanking the objective site, the annealing temperature in the PCR reaction and the reaction environments [37]. The primer pair used to be F27/R1492-amplified DNA, consistent with Edwards et al. [22].

The alignment of the 16S rRNA nucleotide sequence of SAH2_CWMSG comprised 1268 bp. The 16S rRNA stated gene sequence was coordinated in the gene bank database through the NCBI BLAST (http://www.ncbi.nlm.nih.gov). A correlation between the 16S rRNA sequence of this strain and those individuals in the genomic database bank was accomplished. This correlation demonstrated an extraordinary level of sequence similarity (99%) with Streptomyces spp.

The phylogenetic tree ([Figure 3]) resulted from the distance matrices by the neighbor-joining approach and directed by MEGA6 [24]. The analysis included 16 nucleotide sequences. All sites in the missing gaps and misplaced information were removed. In summary, the phylogenetic investigation combined with an ordinary scheme of SAH2_CWMSG demonstrated that the nearest strain is S. rochei strain FMA-91. Thus, S. rochei SAH2_CWMSG was suggested as its name.
Figure 3 Neighbor-joining tree based on 16S rRNA gene sequences showing relationship between Streptomyces rochei SAH2_CWMSG and 19 isolates and closely related type strains of the genus Streptomyces. Only bootstrap values above 50% (percentages of 500 replications) are indicated. Bar, 0.01 nucleotide substitutions per site.

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Nucleotide sequence Gen Bank ID

The nucleotide sequence of the 16S rRNA gene of SAH2_CWMSG strain has been submitted in Gen Bank underneath the accession number ID: KU720627.

Production of L-GLUNase by submerged fermentation

Submerged fermentation is the regularly used process for L-GLUNase production by numerous microbial strains [38],[39]. In this current work, SAH2_CWMSG strain produced L-GLUNase activity of 58 U/ml/min in a specific glutamine medium under the shake flask submerged fermentation on 28°C at 120 h and 200 rpm. These results were better than those stated by Sivakumar et al. [15] who showed that the maximum L-GLUNase production of 17.5 IU was obtained from S. rimosis culture. As well as, they are greater than the results acquired by Mousumi and Dayanand [4] who exposed that the maximum L-GLUNase production (31.55±0.020 IU) was achieved in a batch bioprocess with all optimized conditions under submerged fermentation from Streptomyces enissocaesilis culture.

Extraction and purification of L-glutaminase

Ammonium sulfate fractionation

L-GLUNase produced by SAH2_CWMSG strain was purified using ammonium sulphate. The best fractions (6–12) yield 75% with respect to the crude enzyme ([Table 3]). At ammonium sulphate 75% saturation, the maximum total activity of L-GLUNase (314.166 U), specific activity (0.6 U/mg), yield (92.86%), and the purification folds (0.58) of the purified enzyme were recorded.
Table 3 Effect of different concentrations of ammonium sulfate on L-GLUNase activity, protein content, and specific activity

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The former conclusions matched with that revealed by Balagurunathan et al. [40] who established that at 80% ammonium sulfate saturation, maximum yield of L-GLUNase was produced by Streptomyces olivochromogenes. Moreover, Abdallah et al. [41] reported that at the same ammonium sulphate saturation, the maximum total activity of the L-GLUNase (122.3 U/ml), specific activity (9.7 U/mg), and yield (6.6%) from Streptomyces avermitilis were obtained with purification fold of 2.0.

Absorbance at 280 nm indicated two peaks as shown in [Figure 4] at fractions 1–5 and 6–12. Enzyme activity estimation showed high L-GLUNase activity at peak no. 2 (fractions 6–12) only.
Figure 4 Elution profile of preparations obtained from Streptomyces rochei SAH2_CWMSG by ammonium sulphate (80% saturation) using Sephadex G-100 gel filtration chromatography.

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Physiochemical properties of L-glutaminase

The properties analyzed involved effect of temperature (30–55°C), thermal stability (40–80°C), pH (5–9), pH stability, effect of substrate concentration (L-glutamine: 1–10 mmol/l), reaction time, kinetic determination, metal ions, and other reagents on enzyme activity.

Optimum reaction temperature and thermal stability

The relation between purified L-GLUNase activity and reaction temperatures (30–55°C) was carried out. Maximum enzyme activity produced by SAH2_CWMSG strain was achieved at 40°C ([Figure 5]a), after which a gradual decrease in enzyme activity was noticed by increasing the reaction temperature, and almost 61% of the activity was lost at 55°C. The results obtained were in accordance with Abdallah et al. [41] and Desai et al. [42] who mentioned that L-GLUNases from S. avermitilis and Streptomyces spp. showed optimal activities at temperatures ranged from 30 to 40°C. On the contrary, Iwasa et al. [43] reported that L-GLUNase from Cryptococcus albidus ATCC 20293 exhibited maximal yield at 70°C.
Figure 5 Effect of different temperature (a) and thermal stability (b) on L-GLUNase activity produced by Streptomyces rochei SAH2_CWMSG. The results are presented in enzyme activity.

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The residual activity of L-GLUNase enzyme was determined at different temperatures (40–80°C) for different periods of time (15–60 min), and the relative activity was calculated. At 40°C, enzyme activity (99%) was very stable after 60 min, whereas at 50°C, the enzyme loses 20 and 27% of its activity when incubated for 30 and 45 min, respectively. At 80°C, it retains only 40% of its activity after incubating for 15 min and completely loses its activity after 30 min ([Figure 5]b). Koibuchi et al. [44] mentioned that L-GLUNase from Aspergillus oryzae was stable up to 45°C and loses its activity completely at 55°C. Singh and Banik [45] found that the purified L-GLUNase from Bacillus cereus MTCC 1305 retained 50% and 20% of its stability at 50 and 55°C, respectively, after 30 min of incubation time. In all cases, we noticed that the enzyme activity decreased by increasing the incubation temperature and incubation period.

Optimum reaction pH and stability

The effect of the pH level of the reaction mixture on L-GLUNase was investigated via different buffers. The results revealed that the maximum activity was obtained at pH 7.5 using Na-citrate buffer ([Figure 6]a). This is in accordance with Moriguchi et al. [46] and Rashmi et al. [47] who stated that the pH 7.0 was optimal for the L-GLUNase by the Micrococcus luteus, whereas Iwasa et al. [43] described that pH 6.0 was the optimum in case of Cryptococcus albidus.
Figure 6 Effect of different initial pH (a), different buffers (b) and reaction time (c) on L-GLUNase activity produced by Streptomyces rochei SAH2_CWMSG. The results are presented in enzyme activity.

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Results in [Figure 6]b clearly reveal that L-GLUNase stability increased by increasing the pH, reaching its highest stability at pH 7.5 followed by pH 7.8, after incubation overnight at 4°C. At pH 8, the enzyme retained 69% of its activity when incubated overnight. These results coincide with Roberts et al. [48] who achieved the highest stability of L-GLUNase from Pseudomonas aeruginosa at pH 7.5. Alternatively, Weingand-Ziadé et al. [49] denoted that L-GLUNase showed highest stability at pH 7.0 from Lactobacillus rhamnosus.

Reaction time for enzyme activity

The results in [Figure 6]c showed that the maximum glutaminase activity of 52 U/ml was achieved by incubating the enzyme with glutamine substrate for 30 min at 40°C. The increasing of the incubation period was correlated with decreasing enzyme activity in a gradual manner. These results were in agreement with Fifi [16] who indicated that the pure L-GLUNase showed a maximal activity, in contrast to L-glutamine when incubated at pH 8.0 on 40°C for 30 min.

Calculation of Km and Vmax values for L-GLUNase

L-GLUNase activity increased with increasing of l-glutamine concentration, reaching its maximum velocity. The Km of 1.314 mmol/l and Vmax of 95.24 μMol/min values were anticipated from Lineweaver–Burk plots by using equation derived from non-linear regression analysis of the curve as shown in [Figure 7]. These results showed lower Km and higher Vmax, which indicates the high affinity of L-GLUNase towards its specific L-glutamine substrate. Our results were in agreement with Singh and Banik [45] who found that the Vmax of pure L-GLUNase by Bacillus cereus MTCC 1305 to be 100 µmol/min/ml. On the contrary, our results contradicted with that of Bϋlbϋl and Karakuş [50] who mentioned that the Km and Vmax values were 0.49 mm and 13.86 U/l, respectively, for L-GLUNase from Hypocrea jecorina. Our results indicated that 70 mmol/l was the optimal glutamine concentration for maximum L-GLUNase activity of 95.24 μMol/min. In agreement with these results, Huertá-Saquero et al. [51] reported 80 mmol/l was the optimal glutamine concentration for maximum glutaminase activity from Rhizobium etli.
Figure 7 Lineweaver–Burk (double-reciprocal) plot of 1/V against 1/S giving intercepts at 1/Vmax and −1/Km of L-GLUNase activity produced by Streptomyces rochei SAH2_CWMSG.

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Molecular weight determination of L-GLUNase by SDS-PAGE electrophoresis

The purified enzyme indicated a single band on SDS-PAGE as shown in [Figure 8], and the molecular weight of L-GLUNase was determined to be 55 kDa. These results were in accordance with several authors such as Abdallah et al. [41] and Bazaraa et al. [52], who denoted that the purified L-GLUNase from S. avermitilis and Aspergillus oryzae NRRL 32567 exhibited a single band on SDS-PAGE, and the molecular weight was estimated to be 50 and 68 kDa, respectively. In contrast, Wakayama et al. [53] and Jeon et al. [54] denoted that the molecular weight of purified L-GLUNase from Micrococcus luteus K-3 and Lactobacillus reuteri were 48 and 70 KDa, respectively. The variation of L-GLUNases Mecular weight from diverse sources proposed that it is microorganism specific.
Figure 8 SDS-PAGE analysis of L-GLUNase produced by Streptomyces rochei SAH2_CWMSG. Lane 1, dialyzed enzyme concentrate of ammonium sulfate.

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Effects of metal ions and some compounds on L-GLUNase

The effect of metal ions and some compounds on L-GLUNase were tested, and the results were illustrated in [Figure 9]. The enzyme activity was partially inhibited by all metal ions used, except Mn+2, which activated it. These outcomes were in accordance with those of Yulianti et al. [55] and Dubey et al. [56] who mentioned the positive effect of Mn+2 on L-GLUNase, whereas the addition of other metal ions, for example, Zn+2, Fe+3 and Ca+2, decreased the enzyme activity. On the contrary, Jeon et al. [54] noted that iodoacetate inhibited L-GLUNase activity produced by Lactobacillus reuteri KCTC3594.
Figure 9 The effect of various metal ions and other reagents on L-GLUNase activity produced by Streptomyces rochei SAH2_CWMSG. The results are presented in enzyme activity.

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Biological activities

Antimicrobial activity screening of SAH2_CWMSG isolate

SAH2_CWMSG isolate did not show any activities against bacterial strains used, but showed antifungal activity against all tested fungi as Fusarium solani, A. niger, Candida albicans, Saccharomyces cervecia, Fusarium oxysporium, and Alternaria spp., with inhibitory diameter zone going from 13 to 20 mm, as displayed in [Table 4].
Table 4 Antimicrobial activity (IZ mm) of L-GLUNase activity produced by Streptomyces rochei SAH2_CWMSG

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In vitro antiproliferative activity of L-GLUNase

The anticancer activity of L-GLUNase was verified in contradiction of four kinds of human cancer cell lines by MTT assay in vitro. The results in [Table 5] showed that the pure enzyme has significant efficiency against Hep-G2 cell (100%), MCF-7 cells (97.5%), and HCT-116 cell (100%) at 1000 μg/ml with IC50 of 279.7, 405.1, and 354.2 μg/ml, respectively. Unfortunately, the pure enzyme exhibited the same toxicity to the normal epithelium retina cell line (RPE-1), but still the IC50 was greater than IC50 of HEPG-2 cell line.
Table 5 The cytotoxic effect of SAH2_CWMSG strain L-glutaminase on 4 human tumor cell lines using MTT assay exposed to different concentrations of the drug for 48 h

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These results were in accordance with Nathiya et al. [3] who mentioned that L-GLUNase purified from a bacterium was able to stop a breast carcinoma with IC50 of 256 μg/ml. Similarly, Fifi [16] indicated that L-GLUNase has a noteworthy efficiency contrary to Hep-G2 cell (IC50, 6.8 μg/ml) and a reasonable cytotoxic result against HCT-116 cell (IC50, 64.7 μg/ml). The obtained outcome demonstrated the cytotoxicity of L-GLUNase counter to two cell lines and also showed that the toxic effect was dose response. The IC50 results stated have been assumed in more experimentation.

Antioxidant activity

The antioxidant properties are very vital owing to the deleterious role of free radicals in biological systems. DPPH assay is the most commonly used spectrophotometric procedures for determination of the antioxidant ability of plant extracts and pure compounds owing to its simple, fast, sensitive, and reproducible approach [57]. The IC50 value of L-GLUNase is 165 mg/ml, whereas the IC50 of l-ascorbic acid is 0.65 mg/ml ([Figure 10]a and b). The enzyme was found to scavenge free radicals produced in vitro, and these findings showed L-GLUNase as an antioxidant agent. These outcomes were in agreement with Liyana-Pathirana and Shahidi [58], who revealed that L-GLUNase can scavenge radicals produced in vitro by DPPH assay demonstrating L-GLUNase as antioxidant. Our studies revealed that the scavenging activity increases with the increasing of L-GLUNase concentration.
Figure 10 Antioxidant activity for L-glutaminase from Streptomyces rochei SAH2_CWMSG using DPPH assay.

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  Conclusion Top


L-GLUNase produced from the potent S. rochei SAH2_CWMSG (Gen Bank ID: KU720627) holds proper features in comparison with others formerly described in literature. It is active and steady over a widespread range of pH and temperatures. L-glutamine represented the best substrate for enzyme activity. SAH2_CWMSG strain proved to be an appropriate source of L-GLUNase, which has antifungal, anticancer and antioxidant activities and could, therefore, be potentially used as enzyme supplement which has many applications in industrial and pharmaceutical fields. Further studies will be conducted in our laboratory for maximal optimization and scaling up its production.

Acknowledgements

The authors are thankful to the National Research Center at Dokki, Giza, Egypt, for providing all facilities to support and conduct the present research successfully.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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