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Activity of purified L-glutaminase as a function of the time of the reaction Reaction mixture contained: L-glutamine, 10 μmoles; boric-acid borate, 40 μmoles, pH 8.0; extract protein, 22 μg; total vol.0.5ml; temp, 37°C; reaction time, as indicated.
Source publication
Aim: The aims of the present study were to purify and characterize L-glutaminase from
Penicillium brevicompactum NRC 829; and to evaluate the antitumor activity of the purified
enzyme against different tumor human cell lines.
Study Design: Testing of antitumor activity of L-glutaminase, purified from a filamentous
fungal strain, against four differ...
Context in source publication
Context 1
... and characterization of L-glutaminases from Cryptococcus albidus and C. albidus (ATCC-20293) have been investigated by Iwasa et al. [27] and Ohshita et al. [28], respectively. Purification of L-glutaminase from cell-free extracts of Debaryomyces sp. CECT 11815 was achieved by protamine sulphate treatment followed by anion exchange chromatography and gel filtration as reported by Dura et al. [2]. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) of the enzyme preparation from different purification steps showed that the resolved electrophoretic bands were progressively improved from the crude extract to the final step of the Sephadex G-200 column. It revealed only a single distinctive protein band for the pure preparation of L- glutaminase with an apparent molecular weight of 71 kDa (Fig. 4). There has been wide variation in the molecular weight of L-glutaminases produced from different sources, which could range between 40 and 180 kDa. L-glutaminase with a higher molecular weight (148 kDa) was purified from Ps. aurantiaca by Imada et al. [29]. While 40 kDa molecular weight L-glutaminase by Ps. nitroreducens [30] is the lowest molecular weight recorded for amidase enzyme produced by microorganisms. Most of the L-glutaminases are monomers; however, some of the bacterial glutaminases are reported as dimmers and tetramers. In this concern, Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II with the same molecular weight of about 86 kDa [31]. Results obtained indicate that L-glutaminase activity (Fig. 5), increased as the incubation time increased up to 60 min, after which the linearity of the reaction was not presented and only a slight increase in amidase enzyme activity was reported. L-glutaminase is one of the amidases that are generally active and stable at neutral and alkaline pH. Results (Fig. 6) revealed that pH 8.5 was the optimal pH for L-glutaminase activity from P. brevicompactum using boric acid-borate buffer. In agreement with our results, Ohshima et al. [32] reported that, pH 7.0 to 9.0 to be the optimum pH for amidase activity. In this concern, an optimum pH range of 7.5 - 9.0 was found for L-glutaminase produced from Pseudomonas aeruginosa [19]. L-glutaminase from Debaryomyces sp. CECT 11815 and Lactobacillus rhamnosus showed an optimal working at pH of 8.5 and 7.0, respectively [2,33]. The purified L-glutaminase from P. brevicompactum NRC 829 was more stable in alkaline pH than the acidic one; it retains 100% activity at pH 8.5 even after incubation for 24 hrs at 4 ± 1oC (Table 2). In addition, pH from 7.0 to 9.0 seems to be the most suitable pH range for the storage of this enzyme. While, a slight decrease in the amidase enzyme activity was observed when samples were stored at pH 9.5-10. Our results also demonstrated that, L- glutaminase retained about 56 % of its activity after storing at pH 4.0 for 24 ...
Citations
... The primary role of this enzyme is to facilitate the deamidation process of L-glutamine, producing ammonia and L-glutamic acid (Orabi et al., 2019). In addition, L-glutaminase plays a crucial role in the cellular metabolism of both eukaryotes and prokaryotes (Elshafei et al., 2014). It is one of the most important therapeutic enzymes. ...
... The primary role of this enzyme is to facilitate the deamidation process of L-glutamine, producing ammonia and L-glutamic acid (Orabi et al., 2019). In addition, L-glutaminase plays a crucial role in the cellular metabolism of both eukaryotes and prokaryotes (Elshafei et al., 2014). It is one of the most important therapeutic enzymes. ...
L-glutaminase is an enzymatic catalyst that hydrolyzes glutamine,
converting it into L-glutamic acid and ammonia. It has several
biotechnological uses in the medicinal and food sectors. Moreover, its role
in enzyme therapy for cancer treatment, particularly in acute lymphocytic
leukemia, has been widely acknowledged. This study screened marine
sediment-derived actinobacterial isolates for L-glutaminase. The most
promising L-glutaminase producing strain was selected and identified
through 16S rRNA gene sequence analysis as Streptomyces griseorubens
NAHE (OR462786). The maximum L-glutaminase activity (20.777U/ mL)
was detected in the growth medium containing sucrose and glutamine as the
carbon and nitrogen sources, respectively. The Plackett-Burman
experimental design was used to optimize and identify the factors that have
the greatest impact on L-glutaminase production. This design revealed that
the optimized medium increased L-glutaminase activity by 1.47-fold, higher
than that recorded in the case of the basal cultural conditions. The current
study also showed that L-glutaminase produced by adsorbed marine S.
griseorubens NAHE enhanced enzyme activity by 3.31-fold compared to
conventional free cells. Furthermore, the produced enzyme exhibited a
promising antimicrobial activity against Staphylococcus aureus, Fusarium
oxysporium, Candida albicans, and Aspergillus flavus, which indicates its
suitability for numerous therapeutic applications.
... However, l-glutaminase activity has been researched as a therapeutic agent for colorectal cancer treatments, as described in the work by Mostafa et al. (2021), using a strain of marine bacterium, Halomonas meridian. In the study by Elshafei et al. (2014), a l-glutaminase producing strain of Penicillium brevicompactum, isolated in Egypt, showed antiproliferative activity against the human cell line of hepatocellular carcinoma (Hep-G2), with IC 50 value of 63.3 μg mL −1 . ...
Antarctica harbors a microbial diversity still poorly explored and of inestimable biotechnological value. Cold-adapted microorganisms can produce a diverse range of metabolites stable at low temperatures, making these compounds industrially interesting for biotechnological use. The present work investigated the biotechnological potential for antimicrobial and antitumor activity of filamentous fungi and bacteria isolated from marine sediment samples collected at Deception Island, Antarctica. A total of 89 microbial isolates were recovered from marine sediments and submitted to an initial screening for l-glutaminase with antitumoral activity and for antimicrobial metabolites. The isolates Pseudogymnoascus sp. FDG01, Pseudogymnoascus sp. FDG02, and Penicillium sp. FAD33 showed potential antiproliferative action against human pancreatic carcinoma cells while showing no toxic effect on non-tumor cells. The microbial extracts from unidentified three bacteria and four filamentous fungi showed antibacterial activity against at least one tested pathogenic bacterial strain. The isolate FDG01 inhibited four bacterial species, while the isolate FDG01 was active against Micrococcus luteus in the minimal inhibitory concentration of 0.015625 μg mL ⁻¹. The results pave the way for further optimization of enzyme production and characterization of enzymes and metabolites found and reaffirm Antarctic marine environments as a wealthy source of compounds potentially applicable in the healthcare and pharmaceutical industry.
... The concentration of protein at each step was determined [32]. Purified L-glutaminase was resolved on sodium dodecyl sulfate-polyacrylamide gel (10% SDS), and stained [33]. Further, the activity of L-glutaminase was measured according to the reference [34]. ...
The aims of this study were isolation-purification and characterization of L-glutaminase from L. gasseri BRLHM clinical isolates and investigation of its efficiency as an antimicrobial agent against multidrug-resistant P. aeruginosa. The MICs of L-glutaminase and gentamicin reference were evaluated by the well-diffusion method. The biofilm on the IUD contraceptive was visualized using atomic force microscopy (AFM) image analyses. The purified L-glutaminase possessed significant antimicrobial activity against P. aeruginosa isolates (p < 0.05), and the antibiofilm formation activity of the purified L-glutaminase was stronger than the antibiofilm activity of the referral standard drug, gentamicin (P < 0.05), which were checked by the inhibition of the biofilm formation on the IUD contraceptive device. Investigations indicated that L-glutaminase may have a crucial role in future clinical applications.
... Furthermore, glutaminase is already present in mitochondria, but it must be at the level that allows sequential and fast degradation of glutamine (27). Hence, the use of L-glutaminase deprives the tumor cells of l-glutamine and causes selective death of l-glutamine dependant tumor cells (28,29). The glutamine-deprivation therapy with L-glutaminase that hydrolyzes l-glutamine to l-glutamic acid and ammonia, not only selectively inhibits tumor growth by the blocking of the de novo protein synthesis, but also increase in the superoxide level of oxidative stress that promotes the death of the cancer cells (30). ...
Mesophilic bacteria from soil habitat have been reported to produce extracellular L-glutaminase. The present study was carried out to anticancer screening of L-glutamiase producing bacteria (Kurthia gibsonii) from soil sample of cattle feeding site from Satara parisar, Chhatrapati Sambhajinagar. Results showed, among three soil samples of cattle feeding farms, Kurthia gibsonii was isolated. From that exhibited the highest L-glutaminase activity. Moreover, the in vitro cytotoxic activity of L-glutaminase against the (Lymph Node Carcinoma of the Prostate) LNCaP, (an epithelial, human breast cancer cell line) MDA-MB 231 and hepatocellular (HepG-2) carcinoma cell lines at different concentration (0.47, 0.94, 1.88, 3.75, 7.50, 15.00, 30.00 and 60.00 μg/ml) by the MTT assay and compared with the standard Doxrubcin. The antitumor effect against human liver carcinoma cell line revealed that L-glutaminase produced by Kurthia gibsonii showed potent cytotoxic activity of tested cell line in a dose-dependent manner with an LC50 value of 4.1 μg/ml.
... (Ramadan et al. 1964) and by using GRAMnegative bacillus (Roberts and Frankel 1950). Several fungi have been reported to produce this amidohydrolase with antineoplastic action, such as Aspergillus oryzae (Yano et al. 1988), Trichoderma kaningii (Sakhaei and Alemzadeh 2017), Hypocrea jecorina (Bülbül and Karakuş 2013), Penicillium brevicompatum NRC 829 (Elshafei 2014), Mucor circinelloides (Mary et al. 2020) e Monascus purpureus (Dhale et al. 2011). ...
L-glutaminase is a hydrolytic enzyme with wide biotechnological applications. Mostly, these enzymes are employed in the feed industry for flavor enhancement and acrylamide mitigation. Also, L-glutaminase may have antiviral and antineoplastic effects making it a good choice for pharmaceutical applications. In this study, the strain Monascus ruber URM 8542 was identified through classical and molecular taxonomy using partial sequencing of β-tubulin and calmodulin genes. Subsequently, the optimal culture conditions were evaluated by submerged fermentation (L-glutamine 10 g.L− 1) for L-glutaminase excretion. The isolate was identified as M. ruber URM 8542 which showed significant extracellular enzyme production with a yield of 11.4 times in relation to the specific activity of intracellular L-glutaminase. Regarding the optimization experiments, several factors such as L-glutamine concentration, temperature, and pH were compared using a full factorial design (23). The concentrations greater than 1% proved to be significantly better for glutaminase production (R2 = 0.9077). Additionally, the L-glutaminase was optimally active at pH 7.0 and 30 ºC. The L-glutaminase was remarkably stable across an alkaline pH range (7.0–8.0) and had a thermal stability ranging from 30 ºC to 60 ºC for 1 h. Taken together, these findings suggest that the L-glutaminase produced by M. ruber is a promising candidate for pharmacological application, although further studies need to be performed. To the best of our knowledge, this is the first report of L-glutaminase production by Monascus ruber.
... Among them, the Cohnella glutaminase retained 90% of the catalytic activity in a 25% NaCl solution (Mosallatpour et al., 2019). Moreover, there are many reports on the salt-resistant glutaminases from fungi, such as Cryptococcus nodaensis (Sato et al., 1999), A. oryzae RIB40 (Masuo et al., 2005), and Penicillium brevicompactum NRC 829 (Elshafei et al., 2014). Statistical analyses have suggested that abundant glutamic acid residues on the glutaminase surface could explain their salttolerance mechanism (Yoshimune et al., 2006). ...
... Glutaminase catalyzes the production of L-glutamic acid from L-glutamine, which could improve the umami taste of soy sauce. Due to the high salt content (15-18% NaCl) required by soy sauce fermentation, many studies focused on the screening of salt-tolerant glutaminases from bacteria (Wakayama et al., 2005;Kumar et al., 2012;Mosallatpour et al., 2019;Ferreira et al., 2021) and fungi (Sato et al., 1999;Masuo et al., 2005;Elshafei et al., 2014). However, there are very few studies on the sat-tolerance enhancement of glutaminase via molecular modification. ...
Glutaminase (EC 3.5.1.2) can catalyze the deamidation of glutamine, which has been used to improve umami taste in oriental fermented foods. However, a high salt concentration is still a fundamental challenge for glutaminase application, especially in soy sauce production. To improve the salt tolerance of glutaminase, the self-assembling amphiphilic peptides EAK16 and ELK16 were fused to the N-terminus of a mutant (E3C/E55F/D213T) derived from Bacillus subtilis glutaminase, yielding the fusion enzymes EAK16-E3C/E55F/D213T and ELK16-E3C/E55F/D213T, respectively. As ELK16-E3C/E55F/D213T was expressed as insoluble active inclusion bodies, only the purified EAK16-E3C/E55F/D213T was subjected to further analyses. After the incubation with 18% (w/v) NaCl for 200 min, the residual activities of EAK16-E3C/E55F/D213T in a NaCl-free solution reached 43.6%, while E3C/E55F/D213T was completely inactivated. When the enzyme reaction was conducted in the presence of 20% NaCl, the relative activity of EAK16-E3C/E55F/D213T was 0.47-fold higher than that of E3C/E55F/D213T. As protein surface hydrophobicity and protein particle size analysis suggested, oligomerization may play an important role in the salt-tolerance enhancement of the fusions. Furthermore, EAK16-E3C/E55F/D213T achieved a 0.88-fold increase in the titer of glutamic acid in a model system of soy sauce fermentation compared to E3C/E55F/D213T. Therefore, the fusion with self-assembling amphiphilic peptides is an efficient strategy to improve the salt-tolerance of glutaminase.
... Furthermore, glutaminase is already present in mitochondria, but it must be at the level that allows sequential and fast degradation of glutamine (Lukey et al. 2013). Hence, the use of l-glutaminase deprives the tumor cells of l-glutamine and causes selective death of l-glutamine dependant tumor cells (Nathiya et al. 2011;Elshafei et al. 2014). The glutamine-deprivation therapy with l-glutaminase that hydrolyzes l-glutamine to l-glutamic acid and ammonia, not only selectively inhibits tumor growth by the blocking of the de novo protein synthesis, but also increase in the superoxide level of oxidative stress that promotes the death of the cancer cells (Mustafa et al. 2020). ...
Background
Halophiles are an excellent source of enzymes that are not only salt stable, but also can withstand and carry out reaction efficiently under extreme conditions. l -glutaminase has attracted much attention with respect to proposed applications in several fields such as pharmaceuticals and food industries. The aim of the present study was to investigate the anticancer activity of l -glutaminase produced by halophilic bacteria. Various halophilic bacterial strains were screened for extracellular l -glutaminase production. An attempt was made to study the optimization, purification, and characterization of l -glutaminase from Bacillus sp. DV2-37. The antitumor activity of the produced enzyme was also investigated.
Results
The potentiality of 15 halophilic bacterial strains isolated from the marine environment that produced extracellular l -glutaminase was investigated. Bacillus sp. DV2-37 was selected as the most potent strain and optimized for enzyme production. The optimization of fermentation process revealed that the highest enzyme activity (47.12 U/ml) was observed in a medium supplemented with 1% (w/v) glucose as a carbon source, 1% (w/v) peptone as a nitrogen source, 5% (w/v) NaCl, the initial pH was 7.0, at 37 °C, using 20% (v/v) inoculum size after 96 h of incubation. The produced crude enzyme was partially purified by ammonium sulfate precipitation and dialysis. Of the various parameters tested, pH 7, 40 °C, and 5% NaCl were found to be the best for l -glutaminase activity. The enzyme also exhibited high salt and temperature stability. The antitumor effect against human breast (MCF-7), hepatocellular (HepG-2), and colon (HCT-116) carcinoma cell lines revealed that l -glutaminase produced by Bacillus sp. DV2-37 showed potent cytotoxic activity of all the tested cell lines in a dose-dependent manner with an IC 50 value of 3.5, 3.4, and 3.8 µg/ml, respectively.
Conclusions
The present study proved that l -glutaminase produced by marine bacteria holds proper features and it has a high potential to be useful for many therapeutic applications.
... Singh and Banik, (2013) were reported antitumor activity of L-glutaminase produced by Bacillus cereus MTCC1305.They observed the gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC50 value of 82.27 g/ml in the presence of different doses of L-glutaminase enzyme. However, the purified intracellular L-glutaminase from Penicillium brevicompactum NRC829 inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2) with IC50 value of 63.3µg/ml (Elshafei et al, 2014). ...
The Genomic DNA had been extracted from the liver tissue of the experimental rat groups following 15 weeks of treatment with partial purified L-glutaminase enzyme from E.coli in relation with ethylamine. The DNA samples of all the six treatment groups were amplified by PCR using three different primers specific for determining the presence of P53, bax and G3pdh genes and detect the effect of Ethylenimine and L-glutaminase on the animals at the molecular level. The agarose gel electrophoresis technique used to analyze the PCR amplification products. The result revealed the presence of P53 gene in all treatment groups except for diseased group (T2). This finding demonstrates the mutagenic effect of Ethylenimine that lead to mutation at P53 gene sequence, and therapeutic beneficial of L-glutaminase. Also, there is no PCR amplification product which represent Bax gene sequence for T2 and T5 groups which were administered doses of Ethylenimine, indicating that low doses of L-glutaminase failed to prevent the mutagenic effect of Ethylenimine. While, the G3pd genes is presented only in T2 and T5. Finally, analysis the DNA sequences of the PCR amplified products extracted from liver samples of T2 group treated with Ethylenimine was carried out, then the results were compared with NCBI. The expected mutations were found at thirteen locals and there were only three mutated sequences with the DNA of liver samples from T5 group, while the DNA of animals in group (T6) were showed no mutated regions. The results of this novel study make clear the therapeutic effect of L-glutaminase and how suppress the mutagenic and carcinogenic effect of ethylenimine on P53, Bax, and G3pd genes, and its effect was dose dependent..
... In this context, various studies have focused on the production of bio-products from soybean meal [41], while no reports were mentioned for its potential uses for L-glutaminase production. The enzyme purity was increased gradually by ammonium sulfate precipitation, dialysis, and sephadex-200 [42], and it reached a yield and specific activity of 42.05% and 42.63 U/mg, respectively, in accordance with other reports [32,43]. The molecular weight of Halomonas meridiana L-glutaminase (57.0 kDa) estimated by the SDS-PAGE analysis varied depending on the microbial enzyme source-Bacillus subtilis OHEM11 (54.8 kDa) [37], Bacillus cereus LC13 (35.0 kDa) [38], and Streptomyces canarius FR (44.0 kDa) [43]. ...
L-glutaminase is an important anticancer agent that is used extensively worldwide by depriving cancer cells of L-glutamine. The marine bacterium, Halomonas meridian was isolated from the Red Sea and selected as the more active L-glutaminase-producing bacteria. L-glutaminase fermentation was optimized at 36 h, pH 8.0, 37 °C, and 3.0% NaCl, using glucose at 1.5% and soybean meal at 2%. The purified enzyme showed a specific activity of 36.08 U/mg, and the molecular weight was found to be 57 kDa by the SDS-PAGE analysis. The enzyme was highly active at pH 8.0 and 37 °C. The kinetics’ parameters of Km and Vmax were 12.2 × 10−6 M and 121.95 μmol/mL/min, respectively, which reflects a higher affinity for its substrate. The anticancer efficiency of the enzyme showed significant toxic activity toward colorectal adenocarcinoma cells; LS 174 T (IC50 7.0 μg/mL) and HCT 116 (IC50 13.2 μg/mL). A higher incidence of cell death was observed with early apoptosis in HCT 116 than in LS 174 T, whereas late apoptosis was observed in LS 174 T more than in HCT 116. Also, the L-glutaminase induction nuclear fragmentation in HCT 116 was more than that in the LS 174T cells. This is the first report on Halomonas meridiana as an L-glutaminase producer that is used as an anti-colorectal cancer agent.
