Effect of media composition on L-glutaminase production from lagoon Vibrio sp. SFL-2.
Every organism must find its nutrient substances for energy generation and cellular biosynthesis in its environment . Carbon for synthesizing cell components, nitrogen for cell components, metal ions such as [K.sup.+],[Ca.sup.2+], [Mg.sup.2+], and [Fe.sup.2+] for normal growth and known to be cofactors for various enzymes. They also require high level of NaCl for maintenance of the integrity of their cell walls, stability and activity of their enzymes. Chemically defined media were needed for the cultivation of heterotrophs by defining nutritional requirements.
Numerous media were developed, because the nutritional requirements within bacteria vary widely, there were great differences in the chemical compositions of media used in laboratory. The choice of defined or undefined medium was dependent upon its application. The optimum conditions for activity of any one enzyme in vitro are not necessarily optimum for the same enzyme in vivo . So, the components of a medium were decided upon microorganism, cost, purification process, waste treatment, and so forth. Chemically defined media are useful in biochemical or metabolic studies of organisms .
The production of enzymes was influenced by the variety of physical and chemical conditions. Factors affecting the production of L-glutaminase received much attention in recent years because of its great demand in clinical applications and also in food industries. An understanding of the mechanisms of induction of enzyme in micro-organisms is important for designing ways to obtain maximum yield. These include manipulation of the medium constituents and optimization of the physicochemical parameters, which can in turn influence enzyme synthesis and cell yield. Biosynthesis of L-glutaminase by Vibrio sp. SFL-2 was undertaken in order to find out the optimal cultural conditions to get maximum yield.
Materials and Methods
The bacteria Vibrio sp. SFL-2 was isolated from the sediment of Mullipallam lagoon (Lat. 10[degrees]20'19.71"N & Long. 79[degrees]32'53.65"E) through L-glutamine and phenol red incorporated ZoBell's marine media plate assay technique . The strain was identified by biochemical tests  and named on basis of nomenclature as S F L (Sethusamudram Field Laboratory, Vedharanyam, Tamil Nadu, South India). 2.5 ml of 18 hrs old culture was used as inoculum for 25 ml of pre-sterilized media.
25ml of basal media was selected for L-glutaminase production from Vibrio sp. SFL2 were done between the Modified Sea-Water Complex Broth (SWC), Modified Nutrient Broth (NB), Modified ZoBell's Broth (ZB), Minimal-Glutamine Broth (MGB), Mineral Salt Glutamine Media (MSG) and also Modified Thiosulphate Citrate Bile salt Sucrose (TCBS) media. These media in 100ml Erlenmeyer flask were sterilized in an autoclave at 15 Lbs pressure at 121[degrees]C for 15 minutes except TCBS. Then the medium was modified by L-glutamine and inoculated with inoculum in these different media. The inoculated media were incubated with triplicate at 30 [degrees]C for 24hrs in incubator-cum-shaker with control.
The medium described above was taken as a basal medium and the process parameters under study were varied. After optimization of each parameter, it was included in the next study at its optimal level. The initial pH (6.0 - 8.5), incubation temperature (25 - 45[degrees]C), NaCl Concentration (up to 3.5%), inoculums concentration (0.2 - 1.2 ml), amino acids (glutamine, alanine, arginine, asparagine, cysteine, glutamic acid and lysine at 1% w/w), substrate concentration (0.5 - 2.5% at 1%w/w), carbon sources (glucose, sucrose, maltose, galactose, lactose and trisodium citrate at 1% w/w), nitrogen source (sodium nitrate, yeast extract, peptone, urea, casein, ammonium sulphate and ammonium nitrate at 1% w/w), mineral salts (K[H.sub.2]P[O.sub.4] , Ca[Cl.sub.2],KCl, MgS[O.sub.4], Mn[Cl.sub.4] and N[H.sub.2]P[O.sub.4] at 1% w/w) were optimized for the yield of L-glutaminase. After optimization of process parameters, the time course for the yield of L-glutaminase was evaluated under the optimized conditions for the total period of 108 hrs with 12 hrs interval. All the experiments were conducted in triplicate and mean values were plotted.
Enzyme Elution and Assay:
The specific broth was micro-centrifuged at 10,000 rpm at 4[degrees]C for 20 min. in a cooling centrifuge. Supernatant was collected and incubated with 5ml of modified potassium phosphate buffer at 30[degrees]C for 4 hrs and once again centrifuged. After centrifugation, the supernatant was collected and used for assay of proteins and enzymes.
L-glutaminase assay was done by a method proposed by Imada et-al . The reaction mixture contained 0.5ml of isolated enzyme extract, 0.5ml of 0.04M glutamine (pH 5.6) and 0.5ml of 0.5M acetate buffer (pH 5.6) made up to 2ml by double distilled water and incubated at 30[degrees]C for 30 min. The reaction was stopped by adding 0.5ml of 1.5M trichloroacetic acid. The liberated ammonia in the assay mixture was then estimated by adding 0.2ml of Nessler's reagent to 0.1ml of the above mixture with 3.7ml of distilled water. Suitable controls were included in all the assays in which the substrate was added after trichloro acetic acid. This was then incubated at 20[degrees]C for 20 min and extinction at 450 nm was measured in UV- spectrophotometer (SystronicsVisiscan-167, India). The enzyme yield was expressed as International Unit/gram of dry substrate (IU/gds) as proposed by Nagendra Prabhu and Chandrasekaran . ANOVA and Duncan Multiple Range Test (DMRT) were used to find out the significant between the yields.
Results and Discussion
MGB and MSG broth were support the production of L-glutaminase above 130 IU/gds, particularly yield was 168.93 IU/gds in mineral salt glutamine media from Vibrio sp. SFL-2 (Fig.1). Yield was low as 35.33 IU/gds in sea-water complex media because the media composition was lack of carbon source and mineral salts. The yield of L-glutaminase in the different media composition were ranged from modified SWC < modified TCBS < modified NB < modified ZB < MGB < MSG. Eluted unit of L-glutaminase from Vibrio sp. SFL-2 were subjected to ANOVA followed by DMRT (Table.1). Within the media, SWC broth with TCBS < ZB with NB were significant in L-glutaminase production at p < 0.05.
Almost every biological process was pH dependent; a small variation in pH had changed the rate of process. Maintaining the optimal pH was very important operational requirement for maximizing the yield of L-glutaminase production because the bacterial activity and properties of glutaminase to separation were strongly dependent on Ph . L-glutaminase yield was in increasing trend by increased the pH as 6.0 to 8.0 and after that pH, the yield was decreased in pH 8.5. Fig.2 showed the maximum and minimum yield as 170.53 IU/gds and 24.53 IU/gds in 8.0 and 6.0 respectively. pH 7.5 with 8.5 were significant; in pH 8.5, both the growth and L-glutaminase yield were affected. Thus, the optimum pH for both the growth and L-glutaminase production by Vibrio sp. SFL-2 was 8.0, which was nearly to their isolated environmental (lagoon) pH condition.
Incubation temperature influenced the microbial metabolism both with respect to rates at which cellular processes run and the rates at which nutrients are assimilated . On incubated in different temperature (Fig.3) showed the increased trend of yield from 25[degrees]C to 30[degrees]C and after that the yield was decreased from 35[degrees]C to 45[degrees]C. The optimum temperature for the yield of L-glutaminase from Vibrio sp. SFL-2 was 30[degrees]C and the yield was not affected even up to 35[degrees]C. Maximum and minimum of L-glutaminase yield were 170.13 IU/gds in 30[degrees]C and 128.27 IU/gds in 45[degrees]C respectively. In low temperature, yield also low because the proportion of low melting point of fatty acids increased when the temperature decreased [9,10]. Incubation temperature of 25[degrees]C and 40[degrees]C [less than or equal to] 350C and 40[degrees]C [less than or equal to] 30[degrees]C and 35[degrees]C of L-glutaminase yield were significant with each other.
Yield of L-glutaminase was increased, when increased the NaCl concentration upto 3% as maximum as 201.33 IU/gds and it was low in 1% concentration as 107.07 IU/gds (Fig.4). Yield was suddenly decreased, when the concentration was increased above the 3%, so 3% of NaCl concentration was optimum for the production of L-glutaminase from Vibrio sp. SFL-2. The bacteria didn't produce L-glutaminase without the NaCl because the Vibrios were halophilic, the bacteria were unable or try to grow in the low NaCl concentration,  so there was very low L-glutaminase production and also the high concentration of NaCl was also effect the growth of bacteria as cytostatic. Yield of L-glutaminase in NaCl concentration as not significant with each other.
When the inoculum concentration increased from 0.2 ml to 1.2 ml, yield of L-glutaminase also increased when compared to other concentration. Further increased in inoculum concentration did not promote the L-glutaminase yield (Fig.5). L glutaminase yield was high in 1ml of inoculum concentration as 168.93 IU/gds because the reaction was reciprocal to both the bacterial growth and also for the L-glutaminase production. It was low as 48.53 IU/gds in 0.2ml because the concentration was very small to the total volume of media. 0.8 ml, 1.0 ml and 1.2 ml of continuous inoculum concentrations were significant with each other demonstrated that the inoculum concentration from 0.8 ml to 1.2 ml were not affect the L-glutaminase production in media.
Amino acids were common growth factor required for the synthesis of protein as major nitrogen source ; therefore the yield of L-glutaminase was varied, when the amino acid was changed. Even though each and every amino acid was interchanged by other amino acids, the L-glutaminase yield was varied according to the nature of amino acids (Fig.6). Amino acid varied in increasing trend as glutamic acid < lysine < cysteine < alanine < arginine < asparagine < L-glutamine was correlated with L asparaginase optimization from marine Vibrio sp. by Selvakumar . Yield of L-glutaminase from the Vibrio sp. SFL-2 was high as 168.93 IU/gds in control (glutamine) as optimum because the amide nitrogen of glutamine was source of amino groups in wide range of biosynthetic processes and it also frequently involved in protein active or binding sites. It also serves as source of energy and carbon. Its yield was low as 88.8 IU/gds in the presence of glutamic acid. As increasing trend, cysteine and lysine < alanine, arginine and asparagine [less than or equal to] arginine, asparagine and glutamine were significant at p<0.05 with each other, but the glutamine was significant with arginine and asparagines only.
In chemically defined media, addition of amino acid stimulates the growth, although adjustment of concentration was necessary for media optimization. Thus the L-glutaminase yield was increased by increasing the substrate concentration from 0.5% to 1%. After that the yields become reduced due to the increased trend in the substrate concentration from 1.5% to 2.5% (Fig.7). Yield was high as 47.37 IU/gds in 1% and it was low as 28.31 IU/gds in 2.5% of concentration. Therefore 1% of substrate concentration was the saturation level for the high yield of L-glutaminase. Above the optimum concentration, 1.5% and 2% of substrate concentration was significant with each other. It showed that the L-glutaminase production also impact by high concentration of L-glutamine because it blocks several metabolic cycles of bacterial cell and competitive between the cells for L-glutamine in the low concentration was the only reason for the low yield.
Carbohydrates and related compounds are preferential carbon sources for many genera of microbes (Rosalie, 1999). Enzyme production varied depending on carbon source  and the variation in carbon sources can change the production of enzyme from extracellular to cell-associated diauxic growth . Fig.8 showed the effect of carbon source for yield of L-glutaminase from Vibrio sp. SFL-2 was variably changed, when the carbon source changed. Yield of L-glutaminase was high as 170 IU/gds by utilized the glucose as the carbon source, which present in control because the glucose was essential for glycolytic induction that provoke the transition to glycolytic energy production . Thus the glucose supported both the production of enzyme [17,20] and also biomass [21, 24] of bacteria. DMRT significant showed that the glucose can be substitute with maltose and galactose. Three carbon sources incorporated media's L-glutaminase units were significant with each other as three pairs; sucrose and maltose with lactose [less than or equal to] sucrose and maltose with galactose [less than or equal to] maltose and galactose with glucose. Trisodium citrate as the least, it yields only 122.53 IU/gds of L-glutaminase because it act as superior antimicrobial activity . If sucrose used as source, it gave good growth but poor enzyme production . Thus the increased trend in the glutaminase yield showed, when altered the carbon source as trisodium citrate < lactose < sucrose < maltose < galactose < glucose.
Effect of different nitrogen source (Fig.9) showed that the maximum yield was obtained as 170.40 IU/gds in presence of peptone  because the peptone serves as complex carbon source for the metabolic and buffering capacity . Universal ingredient peptone was normally added to media at concentrations of 0.5-1.0% for routine growth  and amino acid supplementation was not required in complex media containing peptone. Peptone was significant with sodium nitrate and yeast extract, so the combinations of sodium nitrate with peptone or yeast extract were increased the enzyme production . Between the sodium nitrate and peptone, there was no much variation in the yield.
Urea incorporated media yield very low L-glutaminase as 78.4 IU/gds because it effect the growth of bacteria . The variation between the nitrogen source showed the increasing trend in the yield of L-glutaminase as urea < casein < ammonium sulphate < ammonium nitrate < yeast extract < sodium nitrate < peptone. As increasing trend, casein with ammonium sulphate [less than or equal to] ammonium sulphate with ammonium nitrate < sodium nitrate and yeast extract with peptone incorporated media's L-glutaminase units were significant with each other in DMRT.
All the living organisms need some inorganic nutrient for their live, which were mineral salts that do not usually contain the element carbon and when it dissolve in water they separate into ions. L-glutaminase yield obtained from Vibrio sp. SFL-2 in the presence of different mineral salts (Fig.10) showed that the maximum yield was 170.13 IU/gds in presence of K[H.sub.2]P[O.sub.4], which presented in the control because it supported both enzyme production [31-34], enhanced the bacterial growth  and it act as reservoir solution for grown of enzyme crystals .
Even though [Mn.sub.2+] was specifically required for synthesis and activity of enzyme, the yield was minimum as 121.87 IU/gds in presence of Mn[Cl.sub.2]. As increasing trend, Ca[Cl.sub.2]with Mn[Cl.sub.2] [less than or equal to] Ca[Cl.sub.2] with KCl [less than or equal to] KCl with MgS[O.sub.4] [less than or equal to] MgS[O.sub.4] and N[H.sub.2]P[O.sub.4] with [KH.sub.2] [PO.sub.4] incorporated medias L-glutaminase units were significant with each other in DMRT. Thus the increasing trend of the mineral salts showed as mangenous chloride (Mn[Cl.sub.2]) < calcium chloride (Ca[Cl.sub.2])<potassium chloride (KCl) < magnesium sulphate (MgS[O.sub.4]) < sodium dihydrogen phosphate (N[H.sub.2]P[O.sub.4]) < potassium dihydrogen phosphate (K[H.sub.2]P[O.sub.4]).
Incubation time was optimized in modified MSG media after small changes done in the basal media of nitrogen source, NaCl concentration and pH. It played the critical role in L-glutaminase production because the yield of glutaminase was specifically based on substrate utilization and generation time of bacteria. Thus the yield was increased randomly when the incubation time was increased up to 96 hrs because the exponential phase produced more glutaminase when compared to stationary phase , after that the yield become low due to the competitive between them for the substrate and pH increased by accumulation of wastes. Yield was high when incubating the culture for 96hrs as 1603.33 IU/gds due to their generation time  and the quantity was low in starting stage as 253.73 IU/gds on 36hrs (Fig.11). 84 hrs with 108 hrs of incubated culture were significant.
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The effect of parameters and composition of MSG media (g/l) for the maximum yield of L-glutaminase from lagoon Vibrio sp. SFL-2 were elucidation by varied the NaCl concentration, alter the nitrogen source and pH as K[H.sub.2]P[O.sub.4]-1, Magnesium sulphate-0.5, Peptone-0.1, Ca[Cl.sub.2]-0.1, Trisodium citrate dihydrate-0.1, Glucose-5, NaCl-30, L-glutamine -10, 50% aged seawater-1000ml, pH-8.0 with the 1 ml of inoculum concentration was incubated at 30[degrees]C for 96 hrs. After incubation, the L-glutaminase was eluted as 1603.33 IU/gds.
We thank to the authority of Annamalai University for providing the facilities and Dredging Corporation of India (DCI); Sethusamudram Corporation Ltd. (SCL) for their sponsorship.
List of Abbreviations: % - Percentage ' - Minutes " - Seconds [greater than or equal to] - Greater or equal > - Greater than [+ or -] - Standard Error [degrees] - Degree [degrees]C - Centigrade ANOVA - ANalysis Of VAriance. Ca[Cl.sub.2] - Calcium chloride DMRT - Duncans Multiple Range Test Fig. - Figure g/l - Gram per Litre gds - Gram of dry substrate hrs - Hours IU - International Unit KCl - Potassium chloride K[H.sub.2]P[O.sub.4] - Potassium dihydrogen phosphate Lat. - Latitude Long. - Longitude M - molar MgS[O.sub.4] - Magnesium sulphate ml - Milli litre Mn[Cl.sub.2] - Mangenous chloride MSG - Mineral Salt Glutamine media MSG - Minimal-Glutamine Broth NaCl - Sodium chloride Na[H.sub.2]P[O.sub.4] - Sodium dihydrogen phosphate NB - Nutrient Broth pH - Negative logarithm of hydrogen ion concentration rpm - rotate per minute sp. - species SWC - Sea-water complex broth TCBS - Thiosulphate Citrate Bile salt Sucrose SFL - Sethusamudram Field Laboratory, Vedharanyam, T.N. India w/w - Weight by weight ZB - Zobell's Broth
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P. Jeya Prakash * (1), E. Poorani (2) and P. Anantharaman (3)
(1) CAS in Marine Biology, Annamalai University, Porto Novo - 608 502, Tamil Nadu, India * Corresponding Author E-mail: firstname.lastname@example.org (2) Assistant Professor, Department of Biotechnology, Anna University, Coimbatore, India E-mail: email@example.com (3) Reader, CAS in Marine Biology, Annamalai University, Porto Novo - 608 502. Tamil Nadu, India E-mail:paraman cas @yahoo.co.in
Table 1: Significance parameters for L-glutaminase production from Vibrio sp. SFL-2 Parameters Components DMRT L-glutaminase activity (IU) [+ or -] Standard Error Media Sea-water 08.833 [+ or -] 0.837 (a) Nutrient 20.433 [+ or -] 0.982 (b) broth Zobell's 22.333 [+ or -] 0.811 (b) media MG broth 34.433 [+ or -] 1.223 (c) MSG media 42.233 [+ or -] 0.869 (d) TCBS 09.433 [+ or -] 1.235 (a) Temperature 25 35.500 [+ or -] 0.681 (b) ([degrees]C) 30 42.533 [+ or -] 1.277 (d) 35 40.300 [+ or -] 0.866 (c)(d) 40 38.200 [+ or -] 1.097 (b)(c) 45 32.067 [+ or -] 0.796 (a) Inoculum 0.2 12.133 [+ or -] 0.688 (a) concentration 0.4 24.567 [+ or -] 2.172 (b) (ml) 0.6 30.500 [+ or -] 1.877 (c) 0.8 40.367 [+ or -] 0.537 (d) 1.0 42.233 [+ or -] 1.000 (d) 1.2 39.033 [+ or -] 0.821 (d) Carbon Glucose 42.500 [+ or -] 0.681 (d) sources Sucrose 38.500 [+ or -] 1.228 (b)(c) Maltose 40.267 [+ or -] 1.132 (b)(c)(d) Galactose 40.833 [+ or -] 0.674 (c)(d) Lactose 37.100 [+ or -] 0.960 (b) Trisodium 30.633 [+ or -] 1.317 (a) citrate Amino acids Glutamine 42.233 [+ or -] 1.219d Alanine 37.333 [+ or -] 1.121 (c) Arginine 38.533 [+ or -] 1.027 (c)(d) Asparagine 40.500 [+ or -] 1.217 (c)(d) Cysteine 32.500 [+ or -] 1.327 (b) Glutamic 22.200 [+ or -] 1.300 (a) acid Lysine 30.400 [+ or -] 1.044 (b) Incubation 36 063.43 [+ or -] 04.49 (a) time (hrs) 48 146.63 [+ or -] 04.62 (b) 60 192.77 [+ or -] 06.62 (c) 72 265.27 [+ or -] 06.97 (d) 84 342.07 [+ or -] 06.76 (e) 96 400.83 [+ or -] 06.49 (f) 108 322.37 [+ or -] 12.99 (e) pH 6.0 06.133 [+ or -] 0.689 (a) 6.5 11.033 [+ or -] 0.690 (b) 7.0 20.433 [+ or -] 1.357 (c) 7.5 36.167 [+ or -] 1.075 (d) 8.0 42.633 [+ or -] 0.779 (e) 8.5 36.733 [+ or -] 0.644 (d) Substrate 0.5 19.800 [+ or -] 1.184 (a) concentration 1.0 47.367 [+ or -] 1.288 (b) (%) 1.5 61.600 [+ or -] 1.193 (c) 2.0 65.000 [+ or -] 2.343 (c) 2.5 70.767 [+ or -] 0.921 (d) NaCl 0 00.000 [+ or -] 0.000 (a) concentration 1.0 26.767 [+ or -] 0.829 (b) (%) 2.0 42.800 [+ or -] 0.755 (d) 2.5 46.767 [+ or -] 0.705 (e) 3.0 50.333 [+ or -] 0.903 (f) 3.5 38.200 [+ or -] 1.250 (c) Mineral salts K[H.sub.2]P[O.sub.4] 42.533 [+ or -] 1.065 (d) Ca[Cl.sub.2] 33.367 [+ or -] 1.602 (a)(b) KCl 35.167 [+ or -] 1.100 (b)(c) MgS[O.sub.4] 38.700 [+ or -] 1.039 (c)(d) Mn[Cl.sub.2] 30.467 [+ or -] 1.083 (a) Na[H.sub.2]P[O.sub.4] 40.133 [+ or -] 1.311 (d) Nitrogen Sodium 42.067 [+ or -] 1.065 (d) Sources nitrate Yeast 40.933 [+ or -] 1.000 (d) extract Peptone 42.600 [+ or -] 1.101 (d) Urea 19.600 [+ or -] 1.473 (a) Casein 28.533 [+ or -] 1.650 (b) Ammonium 31.233 [+ or -] 1.184 (b)(c) sulphate Ammonium 34.967 [+ or -] 1.203c nitrate Values are mean [+ or -] standard error from three replicates in each group, values not sharing a common superscript letter(s) differ significantly at p<0.05.