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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 241-254 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) was inoculated with 3 % of mycelium suspension of and incubated for 24 h at 180 rpm at 30±2 ºC. Analytical determinations Determination of dry cell weight (DCW): Fermentation broth was centrifuged at 8000 rpm for 10 min and biomass was separated. Separated biomass was suspended in 25 mL, 40 % ethanol and kept for extraction for 2 h on a rotary shaker at 30±2 °C. The extract was again centrifuged at 8000 rpm for 15 min and GSH concentration in the supernatant was determined by alloxan method. Biomass obtained after centrifugation was dried on a preweighed filter paper at 100 °C to constant weight for determination of dry cell weight. Estimation of GSH : GSH forms colored compound upon reaction with alloxan so the concentration of GSH can be determined by UV spectrophotometer, at 305 nm. One gram per liter alloxan was prepared in 0.1 M HCI solution. Glycine (0.1 M) and 0.24 M NaHPO 4 –NaH 2 PO 4 buffer (pH 7.6) were prepared in deionized water. The standard curve was prepared accordingly using standard GSH. Each of the standards was added to a cuvette containing 3.5 mL 0.24 M NaHPO 4 –NaH 2 PO 4 buffer (pH 7.6) and 0.5 mL 0.1 M glycine. The reaction was started by addition of 1 mL alloxan solutions and the durations of the reactions were 20 min. Similar reaction was carried out for extract of GSH from fermentation broth, replacing standard GSH (17). Purification of GSH Aqueous two phase system (ATPS): Effect of PEG molecular weight, PEG concentration and ammonium sulphate concentration: Four different ATPS’s systems were studied to find out effect of molecular weight of PEG (1500, 4000, 6000, and 8000), 20 %; ammonium sulphate, 10 %; cell extract, 10 % and deionized water, 60 %. Similarly, to study the effect of PEG 6000 concentration on partitioning of GSH and proteins Parbatsingh Rajpurohit et al 244 five different systems were prepared each containing PEG 6000 at different concentration (5 %, 10 %, 15 %, 20 %, and 25 %), ammonium sulphate 10 %, cell extract 10 % and concentration of deionized water was adjusted to make final volume to 100 %. To find out the effect of ammonium sulphate concentration, four different systems were prepared each containing ammonium sulphate at different concentration (10 %, 12 %, 15 %, 20 %), PEG 6000 20 %, cell extract 10 % and concentration of deionized water was adjusted to make the final volume to 100 %. In all above mention cases, entire system was mixed and phases dispersed by vortex mixer for 1 min at 30±2 °C. Phases were allowed to separate at 30±2 °C for 12 h. Visual estimates of the volumes of top and bottom phases were made. Samples were carefully extracted from the phases and analyzed for GSH content by alloxan method and for protein content by Bradford method. Kd was calculated for each system by the formula given below: Concentration of GSH in top phase Kd = (3) Concentration of GSH in lower phase Ion exchange chromatography Selection of optimal binding pH for GSH using different resins: The optimum binding pH for GSH on different selected resins like Indion CAM-I, Amberlite XAD-16, Indion 830, Amberlite IR 120H was determined. Based on this, a suitable ion exchange resin was selected for further study. The resin (0.5 mL) was added to each of the tubes, and equilibrated to different pH viz., 3, 4, 5, 6, 7 and 8 by washing twice with equilibration buffer (5 mL of equilibration buffer was added to each tube, and kept for 2 h on rocker shaker). Glycine-HCl buffer (100 mM) was used for pH 3, acetate buffer (100 mM) was used for pH 4 and 5, and phosphate buffer (100 mM) was used for pH 6. In each tube, 2 mL of sample
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 241-254 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) was added and were kept for 2 h on rocker shaker for equilibration. The resin was allowed to settle and the supernatant was used to quantify for GSH content. The resin was washed twice with the equilibration buffer. Adsorbed GSH was calculated by subtracting amt obtained in supernatant from the initial sample of the same pH. Thus, bound percentage was calculated using the following formula. Total GSH content of bound % BOUND = X 100 (4) Total GSH content in loaded The pH with maximum binding was binding pH, and that with least binding can be used as elution pH. The resins were regenerated with 0.5 M NaOH and reused. Determination of static binding capacity and further purification by column chromatography: Different dilutions of the filtered sample containing different concentrations of GSH (0.5 to 3.5 mg/mL) were prepared using phosphate buffer of pH 7. These dilutions were loaded to 0.5 mL of Amberlite IR 120 H previously equilibrated with binding buffer at 25±2 °C) and kept for equilibration. After equilibration it was allowed to settle; the supernatant was removed and quantified for GSH. The isotherms obtained by plotting concentrations of GSH adsorbed (q*) as (µg/mL of resin) vs corresponding equilibrium concentration of GSH in the supernatant (C*) (i.e. unadsorbed concentration; µg/mL), this signify the nature of adsorption. The amt of GSH bound to the adsorbent q * 1 * q k d 1 1 = × + * q max C q max was calculated as the difference between the total amt of GSH loaded and that present in the supernatant after 2 h of equilibration. Maximum adsorption capacity qmax was determined from the plot of q* vs C* (isotherm) and the type of isotherm was found by plotting a graph of 1/q* vs 1/C*. Adsorption isotherm equation: Enhanced Production of Glutathione Where k d = Langmuir isotherm constant. Rearranging Eq. (5), we get, (5) (6) 245 Value of k d can be determined from straightline plot of 1/q* against 1/C*. The intercept of such plots on the 1/q* axis is at 1/q max and slope is k d /q max . A batch study was carried out on strong cation exchange resin Amberlite IR 120H packed in column of diameter 1.1 cm, with bed height 5.7 mL. The headspace of the column was filled with buffer completely to avoid any air gap and then equilibrated with acetate buffer (pH 4.0). Supernatant obtained after ammonium sulphate precipitation was mixed with buffer and loaded on the column until exhaustion point. Concentration of GSH to be loaded was calculated according to static binding capacity of the resin. The pH of the sample was kept at 4 to ensure binding of the GSH to the matrix. Volumetric flow rate of 0.5 mL /min was maintained by peristaltic pump. Washing with same equilibrating buffer to remove the unbound or weakly bound was done. Three different elution strategies were employed to elute the adsorbed GSH from Amberlite IR 120H resin, as elution with 1.5 M NaCl, elution by changing the buffer pH and elution with 1 %. Each time volumetric flow rate was maintained at 0.5 mL/min and 3 mL fractions were collected at a time and analyzed for GSH concentration by alloxan method. Results and Discussion Media optimization by one factor at-a-time method Effects of pH: At an initial pH 6.0, maximum production of GSH, 62.18 mg/L (biomass, 5.55
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