L-glutaminase Production Research Articles

L-glutaminase Production from New Halophilic Marine Streptomyces griseorubens NAHE Isolated from Mangrove Sediment, Red Sea, Egypt

L-glutaminase Production from New Halophilic Marine Streptomyces griseorubens NAHE Isolated from Mangrove Sediment, Red Sea, Egypt

Production and Optimization of L-glutaminase from Halophilic Fusarium solani-melongenae Strain CRI 24 under Submerged and Solid State Fermentation

L-glutaminase is a unique enzyme with catalytic activity and the ability to modulate glutamine levels, making it a valuable enzyme with numerous potential applications. L-glutaminase triggers a distinctive reaction by converting L-glutamine into glutamic acid while releasing ammonia concurrently. This enzymatic process holds potential applications across diverse industries, notably in food and pharmaceuticals. The primary objective of the present research was to identify and isolate a fungal strain proficient in L-glutaminase production from soil found in maritime environments. The physical and nutritional conditions were optimized for maximum synthesis of L-glutaminase under solid state fermentation (SSF) and submerged fermentation conditions (SmF). The isolated organism was identified as Fusarium solani-melongenae strain CRI 24 by morphological and 18S rRNA analysis. The optimum carbon source under SmF and SSF was found to be starch (0.2% w/v). Wheat bran as solid substrate among others showed optimum enzyme activity. On the seventh day of incubation, at pH 8.0 and 0.7% L-glutamine concentration under SSF and SmF, the highest enzyme activity was detected. The greatest enzyme activity in SSF was seen at a moisture content of 10%. Fusarium solani-melongenae species produced the enzyme under optimal conditions and 4.20 and 4.73-fold increase (from 0.8 U/mL to 3.61 U/mL and from 0.781 U/mL to 3.69 U/mL) was achieved after optimization in submerged and in solid state fermentation, respectively. The selective isolation and optimization processes described in this work are a promising technique for the industrial production of L-glutaminase and can be applied in the pharmaceutical and food industries.

Role of Nitrogen Source for L-Glutaminase Production from Fungal Strain using through Submerged Fermentation

L-glutaminase has attracted much attention due its wide range of applications in several fields. The L-glutaminase widely used in pharmaceutical and food industries. L-glutaminase is generally regarded as a key enzyme that controls the delicious taste of fermented foods such as soy sauce. L-glutaminase production was carried out by using supplementation of organic and inorganic nitrogen sources such as yeast extract, malt extract, peptone and urea at concentration ranging from 0.25% to 1.25% with increments of 0.25% and also different inorganic nitrogen sources like ammonium sulphate and ammonium chloride at concentration ranging from 0.025% to 0.125% with increments of 0.025%. The malt extract (1%) produced 399.9 IU, were best organic nitrogen source and ammonium sulphate (0.1%) appear to be good inorganic nitrogen source under submerged fermentation process and showed 546 IU. Current study is an exploring step to industrial sector to upscale their L-glutaminase production and it will useful strategy to commercial sector and alternative to old methods

Role of Nitrogen Source for L-Glutaminase Production from Fungal Strain using through Submerged Fermentation

Role of Nitrogen Source for L-Glutaminase Production from Fungal Strain using through Submerged Fermentation

Application of statistical methodology for the optimization of l-glutaminase enzyme production from Streptomyces pseudogriseolus ZHG20 under solid-state fermentation

BackgroundActinomycetes are excellent microbial sources for various chemical structures like enzymes, most of which are used in pharmaceutical and industrial products. Actinomycetes are preferred sources of enzymes due to their high ability to produce extracellular enzymes. l-glutaminase has proven its essential role as a pharmaceutical agent in cancer therapy and an economic agent in the food industry. The current study aimed to screen the potent l-glutaminase producer and optimize the production media for maximum enzyme yield using one factor at a time (OFAT) approach and statistical approaches under solid-state fermentation (SSF). ResultsOut of 20 actinomycetes strains isolated from rhizosphere soil, 5 isolates produced extracellular l-glutaminase. One isolate was chosen as the most potent strain, and identified as Streptomyces pseudogriseolus ZHG20 based on 16S rRNA. The production and optimization process were carried out under SSF, after optimization using OFAT method, the enzyme production increased up to 884.61 U/gds. Further, statistical strategy, response surface methodology (RSM), and central composite design (CCD) were employed for the level optimization of significant media component (p < 0.05), i.e., wheat bran, sesame oil cake, and corn steep liquor which are leading to increase 3.21-fold l-glutaminase production as compared to unoptimized media. ConclusionsThe presented investigation reveals the optimization of various physicochemical parameters using OFAT and RSM-CCD. Statistical approaches proved to be an effective method for increasing the yield of extracellular l-glutaminase from S. pseudogriseolus ZHG20 where l-glutaminase activity increased up to 1297.87 U/gds which is 3.21-fold higher than the unoptimized medium using a mixture of two solid substrates (wheat bran and sesame oil cake) incubated at pH 7.0 for 6 days at 33 °C.

Studies on Characteristic of Extracellular L–Glutaminase and Identification of L-Glutaminase Producing Bacterial Strain from Cattle Feeding Farm of Chhatrapati Sambhajinagar, Maharashtra State, India

L-glutaminase is gaining marked importance due to its potential clinical applications. A variety of microorganisms, including bacteria, yeast, and lamentous fungi, from soil habitat have been reported to produce L-glutaminase. The present investigation was carried out to isolate and screening of L-glutamiase producing bacteria from soil samples of cattle feeding sites. Among twenty three soil samples of cattle feeding farms around Aurangabad, nineteen bacteria’s were isolated. From that one isolate (AGT-19) that showed significant L-glutaminase activity. Morphological characteristics and 16S rRNA sequencing were used for phylogenetic analysis to identified strain AGT-19 as Kurthia gibsonii. Moreover, Kurthia gibsonii was grown in a medium containing 2.5% succinate and 2.5% L-glutamine showed significant activity of L-glutaminase (0.079 U/mg). The optimum conditions for L-glutaminase production were temperature was 39°C and pH was 7.0. The present isolation of K. gibsonii from cattle feeding site indicated that this bacterium is suitably adapted to the environment of excretion and to point of forming a microbiota in the fecal product. It revealed that soil of cattle feeding farm has diverse bacterial strains and its habitat nature allowed the bacteria expressed the protease activity.

Optimization of L-glutaminase production by Monascus ruber URM 8542 isolated from ice cream industrial effluent.

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 10g.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 1h. 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.

Bioprospecting of Marine Fungi from Coastal Karnataka Region as Potential Source of Economically Important Enzyme L-Glutaminase and their Comparative Genomic Study

Marine fungi are important sources of new metabolites including certain enzymes of medical interest due to their enormous capacity to adapt themselves to extreme environments. Living in a highly competitive ecological niche, they produce certain unusual chemical moieties. Marine biological resources are green, abundant, renewable and aid in economic development. The present study investigates the production of L-Glutaminase which is of therapeutic and industrial importance, from marine fungi of coastal Karnataka. Primary screening on agar plates and submerged fermentation in broth was employed for enzyme production. Both marine yeasts (Pichia sp) and filamentous fungal strains (Aspergillus, Penicillium) were found to be efficient producers of L-Glutaminase. Of the 42 isolates, five potential strains were selected through primary screening and Thin Layer Chromatography was performed to confirm the production. Filamentous fungi were identified through morphological and molecular methods as Penicillium and Aspergillus strains with 99-100% similarity. A. foveolatus (MT667385)and A. nidulans (MT667422) were potential producers (1.58U/ml and 1.41IU/ml). The yeast identified was Pichia kudriavzevii (MT667428), which was a moderate producer of Glutaminase and first marine yeast reported for this enzyme production. Neosartorya quadricincta (MT667427) and P. citrinum (MT667426) are also moderate producers. After screening the marine fungi, the isolated strains’ potential to produce L-Glutaminase was confirmed using SDS PAGE, FTIR and Mass analysis. This study emphasizes the necessity of marine fungal culturing and the scope of use of these fungi for further commercial production of L-Glutaminase which would uplift marine economy.

Production, Purification and Characterization of L-glutaminase from Pseudomonas sp. GPB-06

Production, Purification and Characterization of L-glutaminase from Pseudomonas sp. GPB-06

Screening and production of bacterial L-glutaminase as an anticancer agent from different soil environments in Egypt

Screening and production of bacterial L-glutaminase as an anticancer agent from different soil environments in Egypt

Optimization of Process Parameters for L-Glutaminase Production by Pseudomonas Species ALG3

The present study reports the optimization of process parameters for L-glutaminase production by Pseudomonas species ALG3. The L-glutaminase –producing bacterium had already been isolated from soil at a location within Chukwumemeka Odumegwu Ojukwu University, Uli in Anambra state. It was identified using cultural, biochemical and molecular characteristics. Optimization of L-glutaminase production was done by assessing the effect of various parameters using mineral salt medium contained in 250 ml flasks. The parameters included various bivalent metals, pH, surfactants, growth promoters, amino acids and fermentation time. The Results showed that the addition of FeSO4 stimulated optimum L-glutaminase yield of 38.36 U/ml by Pseudomonas species ALG3, while the least production was observed with MnSO4. Enhanced L-glutaminase yield of 30.73 U/ml was recorded at pH 8.0, while the lowest enzyme accumulation was observed at pH 9.0. The highest L-glutaminase accumulation of 82.25 U/ml was achieved with Tween 80, while the lowest yield was recorded with stearic acid. Urea, stimulated maximum L- Glutaminase yield of 43.82 U/ml, while minimum accumulation was observed with peptone. Proline stimulated highest L- glutaminase yield of 42.63U/ml, while the lowest yield was observed with tyrosine. Highest L- glutaminase yield (91.88 U/ml) was observed after 72h, the yield decreased thereafter. The results obtained in the study illustrated that the optimization of process parameters, increased appreciably the L-glutaminase yield of Pseudomonas species ALG3. This suggest that Pseudomonas species can be used for large scale production of L- glutaminase, which can offer great potential for applications in Medicine and food industry.

Recombinant production and characterization of L-glutaminase (glsA) as a promiscuity therapeutic enzyme.

Because of the therapeutical impacts of hydrolytic enzymes in different diseases, in particular malignancies, we aimed to produce a recombinant putative L-glutaminase (GLS ASL-1) from a recently characterized halo-thermotolerant Bacillus sp. SL-1. For this purpose, the glsA gene was identified and efficiently overexpressed in the Origami™ B (DE3) strain. The yield of the purified GLS ASL-1 was ~ 20mg/L, indicating a significant expression of recombinant enzyme in the Origami. The enzyme activity assay revealed a significant hydrolytic effect of the recombinant GLS ASL-1 on L-asparagine (Asn) (i.e., Km 39.8μM, kcat 19.9 S-1) with a minimal affinity for L-glutamine (Gln). The GLS ASL-1 significantly suppressed the growth of leukemic Jurkat cells through apoptosis induction (47.5%) in the IC50 dosage of the enzyme. The GLS ASL-1 could also change the Bax/Bcl2 expression ratio, indicating its apoptotic effect on cancer cells. The in silico analysis was conducted to predict structural features related to the histidine-tag exposure in the N- or C-terminal of the recombinant GLS ASL-1. In addition, molecular docking simulation for substrate specificity revealed a greater binding affinity of Asn to the enzyme binding-site residues than Gln, which was confirmed in experimental proceduresas well. In conclusion, the current study introduced a recombinant GLS ASL-1 with unique functional and structural features, highlighting its potential pharmaceutical and medical importance. GLS ASL-1 represents the first annotated enzyme from Bacillus with prominent asparaginase activity, which can be considered for developing alternative enzymes in therapeutic applications. KEY POINTS: • Hydrolytic enzymes have critical applications in different types of human malignancies. • A recombinant L-glutaminase (GLS ASL-1) was produced from halo-thermotolerant Bacillus sp. SL-1. • GLS ASL-1 displayed a marked hydrolytic activity on L-asparagine compared to the L-glutamine. • GLS ASL-1 with significant substrate promiscuity may be an alternative for developing novel pharmaceuticals.

PURIFICATION AND CHARACTERIZATION OF L-GLUTAMINASE ENZYME FROM SYMBIONT ENDOPHYTIC BACTERIA OF MARINE PLANT Sargassum sp. AS ANTIMICROBIAL ADJUVANTS

Endophytic bacterial symbiont of marine plant seaweed is a potential source of various bioactive compounds as a raw material for drugs. This research aimed to study the antimicrobial potential of L-glutaminase enzyme isolated from seaweed bacterial symbiont collected from Cikoang Island, South Sulawesi Indonesia. The production of L-glutaminase of bacterial symbiont marine plant Sargassum sp was optimum in nutrient broth using a shaker at 37 °C for 3 d. L-glutaminase was obtained from extraction and fractionation with ammonium sulphate at increased saturation levels of 0-20%, 20-40%, 40-60% and 60-80%, continued by dialysis and column chromatography. Antimicrobial activity assessment was conducted by an agar diffusion method using paper discs. Enzyme fractions were produced by purification using DEAE-Cellulose and Sephadex G-100 column chromatography with a purity of 12.23 and 16.95 times, respectively. The enzyme characterization showed that the optimum condition was at pH 8.0 and 37oC, where at this condition, enzyme activity showed a slight increase after the addition of K+ and Na+ ions and however, significant decrease in enzyme activity after the addition of Cu2+ and Fe2+ ions. The results of the antimicrobial activity evaluation revealed that the L-glutaminase of sephadex G-100 fraction had the highest activity against Escherchia coli and Staphylococcus aureus with the inhibition zone of 12.1 ± 0.29 and 17.4 ± 0.21mm, respectively. These results demonstrated that the environmentally friendly bacterial symbiont of the marine plant Sargassum binderi could be a new alternative as a source to produce the antimicrobial L-glutaminase enzyme. This work probably provides an essential finding for further research on the antimicrobial action of protein fraction from bacterial symbiont of marine plant Sargassum sp. or the other species.

Production, characterization, and antitumor efficiency of l-glutaminase from halophilic bacteria

BackgroundHalophiles 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.ResultsThe 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 IC50 value of 3.5, 3.4, and 3.8 µg/ml, respectively.ConclusionsThe 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.

Marine microbial L-glutaminase: from pharmaceutical to food industry.

Deamination of L-glutamine to glutamic acid with the concomitant release of ammonia by the activity of L-glutaminase (L-glutamine amidohydrolase EC 3.5.1.2) is a unique reaction that also finds potential applications in different sectors ranging from therapeutics to food industry. Owing to its cost-effectiveness, rapidity, and compatibility with downstream processes, microbial production of L-glutaminase is preferred over the production by other sources. Marine microorganisms including bacteria, yeasts, and moulds have manifested remarkable capacity to produce L-glutaminase and, therefore, are considered as prospective candidates for large-scale production of this enzyme. The main focus of this article is to provide an overview of L-glutaminase producing marine microorganisms, to discuss strategies used for the lab- and large-scale production of these enzyme and to review the application of L-glutaminase from marine sources so that the future prospects can be understood. KEY POINTS: • L-glutaminase has potential applications in different sectors ranging from therapeutics to food industry • Marine microorganisms are considered as prospective candidates for large-scale production of L-glutaminase • Marine microbial L-glutaminase havegreat potential in therapeutics and in the food industry.

Bioprospecting of the agaricomycete Ganoderma australe GPC191 as novel source for l-asparaginase production

l-Asparaginase is a therapeutically and industrially-competent enzyme, acting predominantly as an anti-neoplastic and anti-cancerous agent. The existing formulations of prokaryotic l-asparaginase are often toxic and contain l-glutaminase and urease residues, thereby increasing the purification steps. Production of l-glutaminase and urease free l-asparaginase is thus desired. In this research, bioprospecting of isolates from the less explored class Agaricomycetes was undertaken for l-asparaginase production. Plate assay (using phenol red and bromothymol blue dyes) was performed followed by estimation of l-asparaginase, l-glutaminase and urease activities by Nesslerization reaction for all the isolates. The isolate displaying the desired enzyme production was subjected to morphological, molecular identification, and phylogenetic analysis with statistical validation using Jukes-Cantor by Neighbour-joining tree of Maximum Likelihood statistical method. Among the isolates, Ganoderma australe GPC191 with significantly high zone index value (5.581 ± 0.045 at 120 h) and enzyme activity (1.57 ± 0.006 U/mL), devoid of l-glutaminase and urease activity was selected. The present study for the first-time reported G. australe as the potential source of l-glutaminase and urease-free l-asparaginase and also is one of the few studies contributing to the literature of G. australe in India. Hence, it can be postulated that it may find its future application in pharmaceutical and food industries.

Production of a bacterial extracellular L-glutaminase possessing high antioxidant capability

Background and objectives L-glutaminase has utmost practical importance in many fields, such as medicine, pharmacy, and some industries as an effective antioxidant, anticancer, flavor enhancer, and used as an analytical reagent in the determination of glutamate and glutamine. The objective of the present article was to formulate the production medium and to pinpoint the proper growth conditions for the most potent microorganism producing highly active glutaminase enzyme. The general properties of the crude enzyme preparation were determined to detect the proper conditions for enzyme activity. Under the specified conditions, the capabilities of the crude L-glutaminase preparation for antimicrobial and antioxidant activities were investigated. Materials and methods A total of 12 recommended microbial strains were screened for highly active L-glutaminase enzyme production. Factors influencing the production of L-glutaminase enzyme were optimized, and the important properties of the crude enzyme were pinpointed. Finally, biological activities of the crude enzyme were investigated as a preliminary index for the validity of the partially purified L-glutaminase form for medical applications. Results and conclusion Among all tested microorganisms, Bacillus subtilis NRRL 1315 was the most potent producer for L-glutaminase enzyme. The maximum glutaminase production was obtained after 48 h of incubation on a rotary shaker (150 rpm) with the medium containing 5 g/l glucose, 0.1 g/l sodium nitrate, and 10 g/l L-glutamine at 37°C and pH 7.5. The important properties of the crude L-glutaminase were duly pinpointed as follows: optimum enzyme protein concentration and substrate concentration were 2 mg/ml and 40 mM, respectively, and optimum reaction pH and temperature were 7.5 and 37°C, respectively. Under the specified conditions, the crude enzyme exhibited considerable 2, 2′-diphenyl-1-picrylhydrazyl radical scavenging activity.

Optimization of L-glutaminase production by Penicillium chrysogenum

Optimization of L-glutaminase production by Penicillium chrysogenum

A permissive approach for optimization of L-glutaminase production using wheat bran as supporting substrate and assessment of its cytotoxic potentialities

Microbial L-glutaminase has considered as one of the most important therapeutic enzymes considering its anticancer or antitumor activity. In this study, one L-glutaminase producing potent fungus was isolated from the coastal soil and identified as Fusarium nelsonii KPJ-2. During parametric optimization, it was noted that wheat bran supported maximum L-glutaminase production than other agro-industrial wastes tested. Solid substrate fermentation was mechanized with optimum pH of 4.0, incubation temperature at 25 °C, inoculums concentration of 2.0% (v/v), substrate concentration of 7.0% (w/v) and moisture of the production media suits at 20.0% (w/v). Statistical optimization using Response Surface Methodology (RSM) was improved the L-glutaminase production by 14.5% (68.93 U/gds) than unoptimized state. The SEM-EDX analysis demonstrated the overgrowth of fungus on wheat bran and utilization of its associated minerals. A comparative cytotoxic effect of the partial purified glutaminase was examined on both cancerous HCT cell and normal Vero cell line. The result clearly demonstrated that L-glutaminase from F. nelsonii KPJ-2 is specifically cytotoxic against cancer cell line with IC50 of 203.95µg/ml, but, non-responsive against normal cell. The newly isolated fungal strain can produce a considerable amount of L-glutaminase utilizing very low-cost substrate and the enzyme have therapeutic value for real life application owing to its anticancer effectiveness.

A new l-glutaminase from Kosakonia sp.: extracellular production, gene identification and structural analysis

A comprehensive research has been carried out on l-glutaminase producers from marine bacteria. Among the evaluated isolates, Kosakonia radicincitans was found to be a l-glutaminase producer from a marine environment, having the highest enzymatic activities. The optimal condition for l-glutaminase production from K. radicincitans was at pH and temperature of 7.0 and 37 °C, respectively. The enzyme was found stable between pH 4 and 8 and its function also maintained at temperatures between 31 and 43 °C after 30-min incubation. Both the amino acid sequence and domain analysis of the l-glutaminase from the Kosakonia strains revealed various similarities and dissimilarities between these amidase enzymes. Sequence analysis and comparison of l-glutaminase from Kosakonia sp. with other l-glutaminase along with structural prediction were able to identify the unique characteristics of the predicted structures and offer a different perspective into their distinguishable properties for industrial use.