Optimization Of Technics Of Separation And Purification Of Glutathione(GSH) From Yeasts

Glutathione (GSH; L-γ-glutamyl-L-cysteinylglycine)is a kind of useful tripetide which is widely distributed in cells in a variety of species and it has a considerable number of important physiological functions by serving as a biological redox agent and as a coenzyme and cofactor. Up to now, the separation and purification of GSH mainly uses Cu-Salt Method and Ion-Exchange Method. It has been reported that there are some kinds of homemade ion exchange resin using to separate and purify GSH, but the associated techniques are not directly suitable for industrial application.After comparing all kinds of homemade ion exchange and adsorption resin we bought, one of them, i.e. thiourea resin, was selected to study mainly and made satisfied results.Basing on these tests, we developed and established a process of separation and purification of GSH which included following cell operations:extraction of GSH, pretreatment of extraction solution, ultra-filtration, condensation, liquid chromatography and crystal. Through optimization, temperature of condensation under vacuum was selected at65℃, and in this condition the recovery of the operation reached92%. When we doubled the concentration of the sample solution to2400mg/L, the effect of desorption was unchanged, but increased the efficiency; the conditions of chromatography were as follows:filling the column with the pretreated100～200mash resin to35cm of column height, then the column was balanced with the0.1mol/LpH3.0(NH4)2HPO4-HCl or pH3.00.2M (NH4)2PO4-HCl buffer. We pumped the sample solution into the column, washed the column with the same buffer, eluted the column at last washed the column with deionized water, the efficiency of elution of two different buffers is corresponding. GSH could be seperated when the column height was higher than20cm. When the column height was higher than31cm, eluting solution used to crystallise can be collected from two GSH eluting peak. Lower than31cm, this section only can be collected from the second GSH peak. The pH of the crystallizable eluting solution containing GSH was always between3.5～4.5, and we can choose detecting the pH of the eluting solution to collect the eluting solution we wanted. The improved conditions of crystallizing:before crystal concentrating the eluting solution containing GSH under vacuum, and the concentration of the GSH multiples between400to500, then naturally falling the temperature of the condensation solution to room temperature, adding isopropyl alcohol to the condensation solution several times to make the volume of isopropyl alcohol occupy60%of total solution, and adjusting pH of the solution to2.82～2.88, adding1%～2%crystal seeds to solution, crystal according to the sequence of falling temperature as follows:keep it under room temperature for0.5hour, and under10℃for8hours, then washing the crystal and desiccating under vacuum. The recovery of the whole process is about45%, and purity of GSH crystal is99%.During the experiment, we found that after sixty times experiment the effect of separation of the thiourea resin became badly, and regenerative resin’s absorbability got worse. Therefore, we researched the way to regenerate the thiourea resin:in static state experiments, thiourea resin by the disposal of acid and alkali had bigger exchange capacity of GSH than others, so the metho d of disposal by the solution of acid and alkali was selected to exam further. We disposed the thiourea resin by NaOH、HCl solution. After the resin adsorbed GSH in its pure solution pH of which was3.50～3.75, GSH was desorbed from resin with3%NaOH solution. The exchange capacity of GSH was above18mg/g, and the ratio of desorption was about95%. Basing on these tests, we established the technics of regenerating thiourea resin.During optimizing the technics of separation and purification of GSH, we studied the absorption mechanism and found that there were hydrophobic interaction and other interaction between GSH and cinnamene thiourea exchange resin, and the interactions were conspiracy.Based on the mature and stable small scale separation technics, we scaled up the experiment in Yizhuang to make this seperation method be suitbale for industrial production. We scaled up the extraction, ultrafitration, centrifugation, consentration and the chromatographic step, and optimized hot water extraction and eluting condition. The yield of extraction is about80%lower than the small scale90%. In the chromatogrphic step, crystallizable eluting solution can not be collected because of magnifying effect and no GSH product can be got.