L-glutaminase Production Research Articles

Immobilization of <i>Pseudomonas</i> <i>sp.</i> KLM9 in Sodium Alginate: A Promising Technique for L-Glutaminase Production

Microbes and process development are gaining considerable importance owing to their exploitation and suitability for the production of the enzymes of therapeutic and industrial significance. In the present study attempts were made to immobilize the Pseudomonas Sp. KLM9 in different supporting matrix to analyze the possible enhancement in the production of L-Glutaminase. The consequences of the study reveal that, sodium alginate at 3.5% concentration was found to be the better supporting matrix than the others. Further, initial pH 7 of the incubation media, incubation temperature of 37°C were supported good yield of L-Glutaminase by immobilized Pseudomonas Sp KLM9. The fermentation period was reduced to 36 hr with immobilized cells. There is moderate increase in the production L-Glutaminase by the immobilized cells than the free cells of Pseudomonas Sp KLM9. Biomass obtained from the 18 hr old culture of Pseudomonas Sp KLM9 was found better to immobilize for the production of L-Glutaminase.

Immobilization of <i>Pseudomonas</i> <i>sp.</i> KLM9 in Sodium Alginate: A Promising Technique for L-Glutaminase Production

Microbes and process development are gaining considerable importance owing to their exploitation and suitability for the production of the enzymes of therapeutic and industrial significance. In the present study attempts were made to immobilize the Pseudomonas Sp. KLM9 in different supporting matrix to analyze the possible enhancement in the production of L-Glutaminase. The consequences of the study reveal that, sodium alginate at 3.5% concentration was found to be the better supporting matrix than the others. Further, initial pH 7 of the incubation media, incubation temperature of 37°C were supported good yield of L-Glutaminase by immobilized Pseudomonas Sp KLM9. The fermentation period was reduced to 36 hr with immobilized cells. There is moderate increase in the production L-Glutaminase by the immobilized cells than the free cells of Pseudomonas Sp KLM9. Biomass obtained from the 18 hr old culture of Pseudomonas Sp KLM9 was found better to immobilize for the production of L-Glutaminase.

ROLE OF CARBON SOURCE FOR THE PRODUCTION OF L-GLUTAMINASE FROM ASPERGILLUS ORYZAE

Microbial glutaminases are more stable than plant and animal counterparts. In addition to it, they have also been detected in mammalian tissues where they are the major enzymes responsible for catabolic breakdown of glutamine. This study were taken up to the enhance the biosynthesis of L-glutaminase by supplementation of carbon sources were employed in range of 0.25% to 1.0%. The carbon source were supplemented are glucose, fructose and lactose. In case of monosaccharides (fructose) the maximum L-glutaminase production of 494.1 IU was observed at 1% and where as disaccharides like lactose yielded maximum L-glutaminase of 343.5 IU.Key words: L-glutaminase, submerged fermentation, fructose, Aspergillus oryzaeÂ

INVESTIGATION ON THE PRODUCTION OF L-GLUTAMINASE FROM PSEUDOMONAS STUTZERI STRAIN UNDER SOLID STATE FERMENTATION USING VARIOUS AGRO RESIDUES

Solid state fermentation was carried out for the production of L-glutaminase by Pseudomonas stutzeri PIMS6 using different agro residues including green gram husk, Bengal gram husk, cattle feed, wheat bran and groundnut oil cake as solid substrates. L-glutaminase has received significant attention in recent years owing to its potential applications in medicine as an anticancer agent, as an efficient anti-retroviral agent and as a biosensor. In food industries it is used as a flavor and aroma enhancing agent. The maximum yield (55.24 U/gds) of L-glutaminase by Pseudomomonas stutzeri PIMS6 was obtained using cattle feed at 75% initial moisture content, initial pH 8.0, supplemented with glucose (1.0%), ammonium sulphate (1.0%),  inoculated with 5% of inoculum and incubated at 37°C for 96 h. Both physico-chemical and nutritional parameters played a significant role in the production of the enzyme L-glutaminase.Keywords: L-glutaminase, Pseudomonas stutzeri PIMS6, Cattle feed, Solid state fermentation.

Production of L-glutaminase and its optimization from a novel marine isolate Vibrio azureus JK-79

L-Glutaminase is an amidohydrolase which is produced by a variety of micro-organisms including bacteria, yeast and fungi. It is currently used in the treatment of acute lymphoblastic leukemia (ALL) and human immunodeficiency virus (HIV). Another potential application of L-glutaminase is as flavor enhancing agent in soy sauce fermentation. Even though L-glutaminase activity was reported in numerous micro-organisms, production of L-glutaminase from marine bacteria is very scanty. With this view, in the present research work, L-glutaminase production pattern was studied under submerged fermentation using novel marine isolate Vibrio azureus strain JK-79 (GenBank Acession Number JQ820323) based on one-factor-at-a-time approach. The maximum yield of enzyme production (247 U/ml) was achieved in a seawater based medium at pH 8, 37°C, 1% inoculum concentration and 2% glutamine concentration for 24 h. The medium when supplemented with carbon source, it improved the enzyme production from 247 to 321 U/ml with 1.5% maltose. Addition of 2% soybean meal also improved the L-glutaminase production (289 U/ml). The above results indicate the scope for production of salt tolerant L-glutaminase using this novel marine bacterial strain. Keywords: L-Glutaminase, Vibrio azureus JK-79, submerged fermentation, optimization of fermentation, one-factor-at-a-time approach . Afr. J. Biotechnol Vol. 12 No. 50

Biosynthesis of L-Glutaminase by Streptomyces Variabilis ASU319 Isolated from Rhizosphere of Triticum Vulgaris

Out of 210 actinomycetes isolates belonged to the genus Streptomyces recovered from rhizospheric soil of Triticum vulgaris, Zea mays and Vicia faba cultivated in Assuit and New Valley Governorates of Egypt, ten isolates were capable of producing high amounts of L-glutaminase enzyme. The most potent L-glutaminase producer isolate was Streptomyces sp. ASU319 recovered from rhizosphere of Triticum vulgaris. The higher producer actinomycete isolate was identified by sequencing of 16S rRNA as Streptomyces variabilis ASU319 and was deposited in the GenBank nucleotide sequence database under accession number KC145278. Factors affecting L-glutaminase production by the Streptomyces variabilis ASU319(KC145278) were examined and the results revealed that the maximum L-glutaminase value was obtained when the isolate cultivated in the production broth medium supplemented by glutamine 8 mg/ml, adjusted at pH 4 and incubated at 35 °C. These results suggest that L-glutaminase-producing Streptomyces variabilis ASU319 could be used as a plant growth promoting rhizobacteria by increasing the ammonia content in the rhizosphere soil across degradation of the agricultural wastes. Also this isolate can be used in both pharmaceutical and food industrial application for L-glutaminase production on commercial scale.

Optimization of cultural conditions using response surface methodology versus artificial neural network and modeling of l-glutaminase production by Bacillus cereus MTCC 1305

Response surface methodology and artificial neural network were used to optimize cultural conditions of L-glutaminase production from Bacillus cereus MTCC 1305. ANN model was superior to RSM model with higher value of coefficient of determination (99.97ANN>97.78RSM), predicted distribution coefficient (0.9992ANN>0.896RSM) and lower value of absolute average deviation (1.17%ANN<18.47%RSM). Optimum cultural conditions predicted by ANN were pH (7.5), fermentation time (40 h), temperature (34°C), inoculum size (2%), inoculum age (10 h) and agitation speed (175 rpm) with a maximum predicted production of L-glutaminase 666.97 U/l which was close to experimental production of L-glutaminase 667.23 U/l at simulated optimum cultural condition. The production of L-glutaminase was enhanced by 1.58-fold after optimization of cultural conditions. Simple kinetic models were developed using Logistic equation for cell growth, Luedeking Piret equation for L-glutaminase production and modified Luedeking Piret equation for glucose utilization indicating that L-glutaminase fermentation is non growth associated process.

Medium optimization for production of L-Glutaminase (EC 3.5.1.2) by Streptomyces griseus under submerged fermentation

L-Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of l-glutamine. In this report medium optimization was conducted through one -factor -at -a -time approach for the submerged production of L-Glutaminase by Streptomyces griseus using different additional carbon, nitrogen, amino acids, mineral salts and was treated with different concentration sodium chloride. A significant influence of medium components (g/l) Galactose 10.0,Yeast extract 10.0,L-Glutamine 10.0,Magnesium sulphate 0.5, KH2PO4 0.5 , K2HPO4 0.5, NaCl 40 on L-Glutaminase production was noted. The applied methodology was validated using this optimized media, the enzyme activity 45 IU/ml in 48h of incubation was obtained.

Partitioning studies of l-glutaminase production by Bacillus cereus MTCC 1305 in different PEG–salt/dextran

Partitioning studies of l-glutaminase production by Bacillus cereus MTCC 1305 was carried out in different PEG–salt/PEG–dextran system. The partitioning value of l-glutaminase increased with increasing molecular weight of PEG from 2000–4000kDa and decreased with higher molecular weight of 6000kDa. Phase system of PEG 4000 (8.5%)/dextran T500 (9.5%) was selected for the extractive fermentative production of l-glutaminase on the basis maximum partition coefficient (1.31). The production of l-glutaminase was found higher in top phase of ATPS (2.09U/ml) than control media (1.42U/ml). Overall production of l-glutaminase (1.83U/ml) was found lower than top phase (2.09U/ml) in ATPS system. The growth profile with short lag phase and higher cell concentration was obtained for ATPS. The partition coefficient of l-glutaminase increased with increase of system pH and temperature and optimum production was obtained at pH 7.5 and temperature 30°C in top phase of PEG 4000/dextran T500 system.

English

L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of lymphoblastic leukemia. This&nbsp;study&nbsp;investigates&nbsp;the production and optimization of&nbsp;extracellular glutaminase&nbsp;enzyme using several agro-industrial residues by&nbsp;Aspergillus flavus&nbsp;KUGF009&nbsp;using SSF (solid state fermentation).Effect of process variables namely substrates, incubation period, temperature, moisture content, initial pH, supplementary carbon and nitrogen sources and metal ions on the production of L-glutaminase&nbsp;was&nbsp;studied and accordingly, optimum conditions&nbsp;were&nbsp;determined.&nbsp;A. flavus&nbsp;KUGF009&nbsp;&nbsp;was cultured in tea dust to produce L-glutaminase. The organism produced high levels of glutaminase under optimized culture conditions on&nbsp;the&nbsp;5th day of incubation at an optimum pH 4.0, temperature 30&deg;C and moisture content 50% by SSF. Enhanced production occurred on&nbsp;the&nbsp;addition of dextrose, yeast extract and MgSO4&nbsp;as nutritional factors. &nbsp; Key words:&nbsp;L-Glutaminase, anti-leukemic agent,&nbsp;Aspergillus flavus, solid state fermentation

L-glutaminase production by Trichoderma koningii under solid-state fermentation

Solid state fermentation was conducted for the production of L-glutaminase by Trichoderma koningii Oud.aggr. using different agro-industrial byproducts inlcuding wheat bran, groundnut residues, rice hulls, soya bean meal, corn steep, sesamum oil cake, cotton seed residues and lentil industrial residues as solid substrates. Wheat bran was the best substrate for induction of L-glutaminase (12.1 U/mg protein) by T. koningii. The maximum productivity (23.2 U/mg protein) and yield (45.0 U/gds) of L-glutaminase by T. koningii occurred using wheat bran of 70% initial moisture content, initial pH 7.0, supplemented with D-glucose (1.0%) and L-glutamine (2.0% w/v), inoculated with 3 ml of 6 day old fungal culture and incubated at 30°C for 7 days. After optimization, the productivity of L-glutaminase by the solid cultures of T. koningii was increased by 2.2 fold regarding to the submerged culture.

L-Glutaminase Production and the Growth of Marine Bacteria

L-Glutaminase Production and the Growth of Marine Bacteria

Screening and Selection of Marine Isolate for l-Glutaminase Production and Media Optimization Using Response Surface Methodology

The current work details the screening of about 400 marine isolates from various marine niches, from which one isolate was finally selected based on the productivity of glutaminase (71.23 U/l). Further, biochemical identification tests and 16S rRNA sequencing identified this isolate to be Providencia sp. This isolate was taken up for further media optimization studies by using one-factor-at-a-time approach and subsequently by response surface methodology. A face centered central composite design was employed to investigate the interactive effects of four variables, viz., concentrations of glucose, methionine, urea, and succinic acid on glutaminase production. A significant influence of urea on glutaminase production was noted. Response surface methodology showed that a medium containing (g/l) glucose 10.0, urea 5.15, methionine 3.5, succinic acid 6.0, ammonium sulfate 2.5, and yeast extract 6.0 to be optimum for the production of glutaminase. The applied methodology was validated using this optimized media and enzyme activity 119 +/- 0.12 U/l and specific activity of 0.63 U/mg protein after 28 h of incubation at 25 degrees C was obtained.

Continuous production of l-glutaminase by an immobilized marine Pseudomonas sp BTMS-51 in a packed bed reactor

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.

Extra-cellular l-glutaminase production by Zygosaccharomyces rouxii under solid-state fermentation

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.

Continuous production of extracellular L-glutaminase by Ca-alginate-immobilized marine Beauveria bassiana BTMF S-10 in packed-bed reactor.

L-Glutamine amidohydrolase (L-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on L-glutaminase. In this article, we report the continuous production of extracellular L-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl2 for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for L-glutaminase production were incorporated into the continuous production studies. Beads with 12 x 10(8) spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of L-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mL x h), which indicates that continuous production of the enzyme by Ca-alginate-immobilized spores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of L-glutaminase.

L-Glutaminase production by marine Beauveria sp. under solid state fermentation

Extracellular l-glutaminase production by Beauveria sp., isolated from marine sediment, was observed during solid state fermentation using polystyrene as an inert support. Maximal enzyme production (49.89 U/ml) occurred at pH 9.0, 27°C, in a seawater based medium supplemented with l-glutamine (0.25% w/v) as substrate and d-glucose (0.5% w/v) as additional carbon source, after 96 h of incubation. Enzyme production was growth associated. Results indicate scope for production of salt tolerant l-glutaminase using this marine fungus.

Extracellular production of L-glutaminase by alkalophilic Beauveria bassiana BTMF S10 isolated from marine sediment

Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L-glutaminase yield (46.9 U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 °C after 108 h. This enzyme was inducible and growth-associated.

Impact of process parameters on l-glutaminase production by marine Vibrio costicola in solid state fermentation using polystyrene as an inert support

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/m 2) was recovered from the polystyrene SSF system.

Extracellular L-glutaminase production by marine bacteria

Four species of bacteria which includedPseudomonasfluorescens,Vibriocholerae andVibriocosticola were observed to produce glutaminase both as extracellular and intracellular fractions. Comparatively both the fractions were higher in mineral media supplemented with 1% glutamine than in nutrient broth added with or without glutamine. Extracellular glutaminase production was about 2.6–6.8 times greater than the intracellular production by all the tested strains.