d(GC) - Association of Biotechnology and Pharmacy

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Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 241-254 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) GSH production when added at concentration of 10 mM. Methionine is a precursor for cysteine biosynthesis and hence could have a positive effect on GSH production. Tyrosine and lysine showed inhibitory effect on intracellular GSH content. Wen et al. (24) studied utilization of amino acids to enhance GSH production in S. cerevisiae T65 and reported cysteine to be the most important amino acid, which increased intracellular GSH content greatly but inhibited cell growth at the same time. Methionine, glycine, serine, and glutamic acid were also reported to have positive effect on GSH production. Cha et al. (21) observed similar effect amino acid 252 addition on GSH production using S. cerevisiae FF-8. Purification of GSH Aqueous Two Phase System : Effect of PEG molecular weight, PEG concentration and ammonium sulphate concentration: The effect of molecular weight of PEG on partitioning of GSH and protein was studied. The partitioning of GSH in top phase increased as molecular weight of PEG was increased. Maximum Kd (3.66) was achieved with PEG 6000 followed by with PEG 1500 (2.48) and PEG 4000 (2.85). With PEG 8000 Kd value was 2.27 Fig. 7. Selection of optimum binding pH for GSH on different resins Fig. 8. Langmuir adsorption isotherm of GSH using Amberlite IR 120H Parbatsingh Rajpurohit et al
Current Trends in Biotechnology and Pharmacy Vol. 6 (2) 241-254 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online) indicating that GSH was partitioned in both phases. Also percent protein in top phase was 38.23 % indicating that remaining proteins were in bottom phase with PEG 8000. Total percent protein in top phase was found to be (53.48 %) with PEG 8000 which was undesirable. Results suggested that with use of PEG 6000 around 61.87 % unwanted proteins could be separated from GSH cell extract in a single step. Hence, PEG 6000 was selected for further studies. The effect of concentration of PEG on partitioning of GSH and protein was further taken in to consideration. As partitioning of GSH in top phase increased as concentration of PEG was increased. Maximum Kd (3.55) was achieved at PEG 6000 concentration of 20 % which decreased to 3.1 with further increase in PEG concentration. This could be due to increased viscosity of the system at high PEG concentration which in turn causes resistance to mass transfer. The percent protein in top phase was found minimum (38.36 %). The effect of ammonium sulphate concentration on partitioning of GSH and protein was also studied. At 12 % ammonium sulphate concentration maximum Kd (3.7) was achieved. The percent proteins in top phase were 38.5 % indicating that remaining were separated from GSH cell extract. At low salt concentrations percent proteins in top phase were less which may be due to solubilization of proteins at low salt concentration (salting in effect). At high salt concentration percent protein in top phase were found to be increased which may be because of the fact that proteins tend to precipitate and are retained in upper phase. Ion exchange chromatography Selection of optimal binding pH for GSH on different resins: The percentages of GSH bound on various matrices at different pH are shown in Fig. 7. The adsorption of GSH was found to be maximum on Amberlite IR 120H (at pH 4.0, 74.39 %), Indion CAM (at pH 3.0, 65.34 %), Amberlite XAD-16 (at pH 3.0, 52.14 %) and Indion 830 (at pH 5.0, 45.60 %). Since maximum binding was on Amberlite IR 120H resin at pH Enhanced Production of Glutathione 253 4.0 it was selected for further studies. Determination of static binding capacity and further purification by column chromatography: Equilibrium adsorption isotherm was studied to find out adsorption isotherm pattern for purification of GSH using Amberlite IR 120H resin. The adsorption isotherm data was used to determine the adsorption capacity of the matrix. The GSH adsorption followed a typical Langmuir type of isotherm as shown in Fig. 8 for Amberlite IR 120H resin. The maximum capacity of the matrix (q ) for GSH was found to be 3.081 mg/ max mL. A plot of 1/q* Vs 1/C* (Fig. 9) gave a linear correlation confirming the adsorption to be of the Langmuir type. Lower elution efficiency with 1.5 M NaCl (8.64 %) and 100 mM phosphate buffer of pH 8 (5.91 %) was observed (Table 5). Elution with 1 % H SO showed the best elution pattern. 2 4 Percent elution was very high (89.19 %) as compared to salt and pH elution. Conclusions Optimization of the fermentation medium could increase the GSH production by S. cerevisiae NCIM 3454 from 55.28 mg/L to 148.45 mg/L. L-cysteine acted as stimulator in production of glutathione (163.12 mg/L). Aqueous two phase system was found to be good alternative during purification of GSH prior to adsorption chromatography. System containing PEG 20 %, and ammonium sulphate 12 % gave maximum protein recovery of GSH in top phase and separating maximum proteins in bottom phase. Amberlite IR 120H showed maximum GSH adsorption (74.39 %) at pH 4 and maximum elution was achieved with 1 % H SO 2 4. Acknowledgement Authors are thankful to the Department of Biotechnology, Ministry of Science and Technology, India, for providing financial assistance during the course of this research. References 1 Anderson, M.E. and Meister, A. (1983). Glutathione. Annu Rev Biochem. 52: 711- 760.
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