Study On The Permeability And Capability For GSH Synthesis With Yeast Cells

Glutathione(GSH), as an active tripeptide containing ?-glutamyl and thiol, can be biosynthesized from glutamic acid, cysteine and glycine catalyzed consecutively by ?-glutamyl-cysteine synthase( ?-GCS) and glutathione synthase(GS). GSH plays many important physiological roles in living cells, such as antioxidant,detoxification. As a result, GSH is widely used in food, medicine, health products and other fields. At present, the production of GSH mainly by fermentation method and enzyme method.GSH is an intracellular product, accumulated to a certain concentration will produce the feedback inhibition to GSH synthetase. In this paper, flocculation yeast SP5, a strain that generally possesses the ability of synthesis and storage of GSH, was used for GSH production. In order to accumulate GSH in intracellular and extracellular form at the same time, reduce or relieve feedback inhibition, improve the catalytic efficiency of GSH synthetase and the total yield of GSH, the effect of low pH stress, freezing and thawing, dry, osmotic shock, ultrasound, organic solvent and surfactant treatment on the membrane permeability of yeast cells was studied. The main results of this dissertation are as follows:(1) In the natural conditions of flocculation yeast SP5 fermentation to 24 h, cell dry weight to 7.49 g/L, the GSH produced mainly accumulated in cells(about 7.2mg/g stem cells), the total yield of GSH was 53.9 mg/L; The accumulation content of GSH in the fermentation liquid is very low, even could'nt be detected.(2) The pH value of fermentation will reduce from 6 to 4.5 in yeast growth process.The content of extracellular GSH reached 35.45 mg/L in pH 1.5, treated 3 hrs,at the same time, the cell dry weight and cell survival rate decrease 20% and 40%respectively. GSH can synthesis and accumulation again by pH control in the process of GSH enzymatic synthesis, the total yield of GSH reached 446 mg/L, which increased by 23% compared with the control 363 mg/L.(3) The content of GSH in yeast cells did not change significantly after treated with freezing and thawing, but were significantly decreased when treated with dry,osmotic shock,or ultrasound. However, the total of GSH by enzymatic synthesis is not increase obviously, some even decreased.(4) The addition of certain concentration acetone, tetrahydrofuran, ethyl acetate,butanol, chloroform, toluene or ethyl ether in fermentation liquid cultured 2.5 h, the content of GSH in yeast cells were significantly decreased. It can accumulate GSH in intracellular and extracellular form at the same time when enzymatic synthesis GSH,the total yield of GSH increased significantly. Acetone is the most effective, 7%(V/V)acetone was added to the enzymatic synthesis reaction solution, enzyme catalyzed synthesis of 48 h, the intracellular, extracellular and total yield of GSH reached,respectivly, 293 mg/L, 258 mg/L and 551 mg/L.(5) The addition of certain concentration SDS?LS?CTAB or Triton X-100 in fermentation liquid can significantly decrease the content of GSH in yeast cells, the higher the concentration, the higher the content of extracellular GSH. It can accumulate GSH in intracellular and extracellular form at the same time when enzymatic synthesis GSH, the total yield of GSH increased significantly. LS is the most effective, 0.5 g/L LS was added to the enzymatic synthesis reaction solution,enzyme catalyzed synthesis of 24 h, the total yield of GSH reached 435 mg/L.(6) The cell membrane permeability of flocculation yeast SP5 has changes in different treatments, flocculation morphology and cell structure have also changes.The secretion and regulation mechanism of GSH in yeast has three possible models,leak model, secretion carrier model and carrier inversion model. Further research is needed in the secretion carrier model.