Purification, Kinetic Properties and Antitumor Activity of L-Glutaminase from Penicillum brevicompactum NRC 829
Abstract and Figures
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 different tumor human cell lines.
Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering
and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between
January 2011 and February 2012.
Methodology: P. brevicompactum NRC 829 was grown and maintained on modified
Czapek Dox agar (MCD) medium. Cell-free extract was directly used as the source of crude
enzyme. L-glutaminase was purified by heat treatment for 20 min at 50ºC, followed by gel
filtration on Sephadex G-100 and G-200 columns.
Results: An intracellular L-glutaminase from Penicillium brevicompactum NRC 829 was
purified to homogeneity (162.75 fold) with an apparent molecular mass (Mr) of 71 kDa. The
purified enzyme showed its maximal activity against L-glutamine when incubated at pH 8.5at 50ºC for 30 min. The purified enzyme retained about 92 % of its initial activity after
incubation at 70ºC for 30 min indicating the thermo-stability nature of this enzyme. The
highest activity was reported towards its natural substrate, L-glutamine, with an apparent
Km value of 1.66 mM. The purified enzyme inhibited the growth of human cell line
hepatocellular carcinoma (Hep-G2), with IC50 value of 63.3μg/ml.
Conclusion: L-glutaminase purified from Penicillium brevicompactum NRC 829 is a
potential candidate in food and pharmaceutical industries.
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... 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.
... 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. ...
... 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.
... 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..
This study was to compare GABase [a mixture of γ-aminobutyric acid (GABA) aminotransferase and succinic semialdehyde dehydrogenase] and glutaminase inhibitory activities of 20 herbal extracts and investigate the isolation, structural elucidation and those inhibitory activities of three acylated flavonol monoglycosides from the selected extract of Laurus nobilis L. (laurel). On the basis of the NMR spectroscopic data and the ESI MS spectra together with the comparison with the literature values, three compounds were identified as kaempferol-3-O-(4″-E-p-coumaroyl)-α-l-rhamnopyranoside (1), kaempferol-3-O-(3″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (2) and kaempferol-3-O-(2″,4″-di-E-p-coumaroyl)-α-l-rhamnopyranoside (3), respectively. The IC50 values of GABase inhibitory activity of 1-3 and p-hydroxybenzaldehyde (HBA) as control were 0.24 mM, 0.14 mM, 0.12 mM and 0.43 mM, respectively. Additionally, the IC50 values of glutaminase inhibitory activity of 1-3 and 6-diazo-5-oxo-l-norleucine (DON) as control were 0.34 mM, 0.13 mM, 0.14 mM and 0.33 mM, respectively. The results suggest that the extract from laurel shows the strongest biological activities among 20 herbal extracts and three acylated flavonol monoglycosides may serve as potential lead compounds for the prevention and treatment of neurodegenerative and lifestyle-related diseases by targeting GABase and glutaminase. This is the first report on GABase and glutaminase inhibitory activities of 1-3.
L-glutaminase (E.C.3.5.2.1) is an antineoplastic enzyme and in the present study, an extracellular L-glutaminase was produced from a marine local strain identified as Pseudomonas sp. RAS123. The enzyme was produced from free cultures and from cultures immobilized on and in different supports. Pseudomonas sp. RAS123 L-glutaminase produced from immobilized cultures was purified to homogeneity. The specific activity of the enzyme reached 698.655 U/mg protein, with Km and Vmax value of 3.2 mg/ml and 2000 U/ml, respectively. A single band with a molecular weight of about 32.0 kDa was produced by the purified enzyme on SDS-PAGE. Further findings indicated that the pure enzyme's maximum activity occurred at 50°C and pH 9. The enzyme was stable at 60°C for 60 min and in the pH range of 8.0 to 10.0, The effect of chemicals showed that Mn2+, Mg2+, Ni2+ and Fe2+activated the enzyme, while SDS (10% w/v) strongly inhibited the activity of the enzyme. The purified enzyme showed cytotoxic activity against HCT-116, HepG2, MCF-7, HeLa, and CCL-86 cell lines tested with IC50 values of 122, 175, 195, 306, and > 500 µg/ml, respectively. Also, the antibacterial effect of the enzyme showed activity against Staphylococcus aureus, Bacillus subtilis, Streptococcus mutants, Enterobacter cloacae and Escherichia coli. These findings demonstrate that L-glutaminase might be used in numerous biotechnological applications, particularly food and pharmaceutical processing.
Actinomycetes from terrestrial and marine ecosystems have long been recognized either as organism of academic curiosity and organism of antibiotic producers. L-glutaminase is an enzyme produced by various microorganisms which are currently used for the treatment of leukemia, as flavor enhancer and also as enzyme sensors. Even though L-glutaminase activity was reported in various microorganisms, L-glutaminase from actinomycetes in general and marine actinomycete in particular is very scanty. From the reviewed literature, information on L-glutaminase production from actinomycetes is very scanty. With this view, marine sediment sample was collected from mangrove rhizosphere of Parangipettai coastal area (Lat. 11 .29' N; Long. 79 .46'E). Totally 20 L-glutaminase oo producing actinomycete strains were isolated by using minimal glutamine agar medium supplemented with substrate 1% L-glutaminase, 0.012% phenol red and 3 % NaCl. All the 20 actinomycete strains were micromorphologically identified as Streptomycetes. L-glutaminase enzyme was produced from all the 20 actinomycete strains by submerged fermentation and tested by semi quantitative assay. Crude L-glutaminase produced from strain P2 produced 38 mm of pink color zone. L-glutaminase produced from strain P2 was purified further by precipitation and dialysis methods. In semi quantitative assay of dialysate showed 45 mm of zone of color change. Optimal condition for L-glutaminase production was determined at pH 7, temperature 30°C and salinity 3.5%. Based on the studied phenotypic characteristics the potential actinomycete strain was identified as Streptomyces olivochromogenes (P2). The selective isolation procedure and semi quantitative assay developed in this present study will be a suitable and best method for therapeutic enzymes in general and L-glutaminase in particular.
(1) Both glutaminases A and B of Pseudomonas aeruginosa are inactivated by urea and guanidine hydrochloride, and the activities are partially restored by removal of the denaturants, while sodium lauryl sulfate denatured irreversibly the isozymes. (2) Glutaminase A consists of 4 identical subunits (mol. wt., 35, 000) and B is composed of one polypeptide chain (mol. wt., 67, 000). (3) Glutaminase A, which catalyzes the hydrolysis and also the hydroxylamino-lysis of L and D isomers of glutamine and asparagine, does not act on γ-N-substituted glutamine e. g., γ-glutamylhydrazide. Some L- and D-γ-glutamyl derivatives, e. g., L- and D-γr-glutamyl-hydrazide, L- and D-γ-glutamylmethylester, and L-γ-glutamyl-L-alanine are substrates for glutaminase B, which does not catalyze the hydrolysis and hydroxylaminolysis of asparagine. α-Amino adipamic acid and α-amino substituted amino acids are inert for both the isozymes. (4) The acylation step is rate-limiting in the catalytic reactions by both the isozymes.
A marine Pseudomonas sp BTMS-51, immobilized by Ca-alginate gel entrapment was used for the production of extracellular l-glutaminase under repeated batch process and continuous process employing a packed bed reactor (PBR). Immobilized cells could produce an average of 25 U/ml of enzyme over 20 cycles of repeated batch operation and did not show any decline in production upon reuse. The enzyme yield correlated well with the biomass content in the beads. Continuous production of the enzyme in PBR was studied at different substrate concentrations and dilution rates. In general, the volumetric productivity increased with increased dilution rate and substrate concentrations and the substrate conversion efficiency declined. The PBR operated under conditions giving maximal substrate conversion efficiency gave an average yield of 21.07 U/ml and an average productivity of 13.49 U/ml/h. The system could be operated for 120 h without any decline in productivity.
Process parameters influencing l-glutaminase production by marine Vibrio costicola in solid state fermentation (SSF) using polystyrene as an inert support were optimised. Maximal enzyme yield (157 U/g drysubstrate) was obtained at 2% (w/w) l-glutamine, 35°C and pH 7·0 after 24h. Maltose and potassium dihydrogen phosphate at 1% (w/w) concentration enhanced enzyme yield by 23 and 18%, respectively, while nitrogen sources had an inhibitory effect. Leachate with high specific activity for glutaminase (4·2 U/mg protein) and low viscosity (0·966 Ns/m2) was recovered from the polystyrene SSF system.
SUMMARY L-Asparaginase and L-glutaminase activities were detected in many micro- organisms and the distribution of these activities was found to be related to the classification of micro-organisms. Among 464 bacteria, the activities occurred in many Gram-negative bacteria and in a few Gram-positive bacteria. Most members of the family Enterobacteri- aceae possessed L-asparaginase. L-Asparaginase and L-glutaminase occurred together in a large proportion of pseudomonads. Among Gram-positive bacteria many strains of Bacillus pumilus showed strong L-asparaginase activity. Amidase activities were also observed in several strains in other families. L-Asparaginase activity was not detected in culture filtrates of 261 strains of species of the genera Streptomyces and Nocardia, but L-asparaginase and L- glutaminase were detected when these organisms were sonicated. The amidase activities in culture filtrates of 4158 fungal strains were tested. All the strains of Fusarium species formed L-asparaginase. Organisms of the genera Hjyomyces and Nectria, which are regarded as the perfect stage of the genus Fusarium, also formed L-asparaginase. Several Penicillium species formed L-asparaginase. Two organisms of the family Moniliaceae formed L-glutaminase together with L-asparaginase, and a few ascomycetous fungi formed L-asparaginase or L-glutaminase. Among I 326 yeasts, L-asparaginase or L-glutaminase occurred frequently in certain serological groups of yeasts : VI (Hansenula) group, Cryptococcus group and Rhodotorula group. Many strains of Sporobolomyces species also showed L-asparaginase activity. Several strains of Cryptococcus and Rhodotorula group possessed L-glutaminase and L-asparaginase. L-Glutaminase alone was formed in many strains of Candida scottii and Cryptococcus albidus, both of which are related to Basidiomycetes.
Solid-state fermentation (SSF) was carried out for the production of extra-cellular l-glutaminase by the saline tolerant yeast Zygosaccharomyces rouxii NRRL-Y 2547 using two agro-industrial substrates, wheat bran and sesamum oil cake, which were selected after a screening of four substrates and two moistening media (sea water and 10% NaCl in tap water). Results showed better efficiency of NaCl medium for wheat bran and seawater for sesamum oil cake. Maximum l-glutaminase titres (7.5 and 11.61 U/g dry substrate- gds in comparison to 2.2 and 2.17 U/gds as initial values for wheat bran and sesamum oil cake, respectively) were produced when SSF was carried out with 64.2% initial moisture content of the substrate, 2 ml of 48 h old inoculum, 30°C as incubation temperature and 48 h of incubation period in both the substrates, which were 3.5- and 5-times higher than the initial production for wheat bran and sesamum oil cake, respectively. External supplementation of fermentation medium with various organic and inorganic nitrogen sources was of no benefit for enzyme production.
Systematic investigations on the effect of varying dissolved oxygen (DO) concentrations and agitation profiles on glutaminase yield from Zygosaccharomyces rouxii NRRL Y-2547 were conducted in a batch stirred tank reactor. The agitation rate was varied in the range of 100–400 rpm, while the DO values studied were 20, 40 and 60 %, respectively. Furthermore, a stepwise reduction in DO was investigated wherein the DO concentration was decreased from 40 % for the first 12 h to 20 % for the following 24 h, and finally decreased to 5 % DO up to 54 h (end of batch period). The maximum enzyme yield in all cases was observed after 48 h at 37 °C. An agitation rate of 200 rpm and a three-stage DO maintenance strategy (40-20-5 % DO) gave maximum glutaminase productivity. Yeast extract and glucose feeding increased the enzyme production from 370.75 ± 4.97 units/L to 437.14 ± 4.44 and 417.99 ± 5.82 units/L, respectively.
Glutaminase activity was found in a water extract of a wheat bran koji (extracellular fraction) of Aspergillus oryzae strains Lee-1, H-16 and MA-27-IM isolated from a commercial koji ssed for soy sauce fermentation, as well as in thier mycelia (intracellular fraction). Both the intracellular and the extracellular glutaminases were purified from strain MA-27-IM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weight of the intracellular and the extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various γ-glutamyl compounds besides l-glutamine but did not exhibit asparaginase activity. Further investigation of these preparations inidicated that these glutaminases possessed almost the same properties, suggesting their similarity.