The Study Of Preparation And Fusion Of Isoflavone Translation Bacterial Protoplast

Soy isoflavones (SI) mainly composed by daidzein, genistein and glycitein are the secondary metabolites formed in the process of growth of soybean. It can play muti-physiological roles including prevention of cancer, reduction of blood fat, prevention of osteoporosis, improvement women's menopausal syndrome etc through binding with estrogen receptors after being absorbed. Soy isoflavones can be transformed into a series of different metabolites under the effect of microbial flora of gastrointestinal tract, such as dihydrodaidzein (DHD), equol and O-desmethylangolensin(O-Dma). Recent study has shown that the metabolites of soy isoflavones have higher and wider biological activity than soy isoflavones themselves.Protoplast fusion technique is a kind of method that can make two different parental cells which has different genetic characteristics to be fused as a new one by physical or chemical methods. Due to the removal of the cell wall, protoplast membrane are prone to fusing,which makes the genome of the two parental cells to be recombinized more easily.Bacteria strain AUH-HM195(Enterococcus hirae) isolated by our lab is a facultative anaerobe. Eventhrough it can grow in aerobic and anaerobic conditions, it can convert daidzein into O-Dma only grow in anaerobic conditions. In order to make bacteria strain AUH-HM195 to be of daidzein biotransforming activity in anaerobic conditions, protoplast fusion between strain AUH-HM195 and aerobic microorganisms were studied in this paper. Both Saccharomyces cerevisiae and Pseudomonas aeruginosa R5 isolated by our lab were chosen as aerobic microorganisms for fusion. The optimal conditions for preparation and regeneration of protoplast of bacteria strain AUH-HM195 were as follows: bacterial age 6h, lysozyme concentration 5mg/mL, enzymolysis time 1h, osmotic pressure stabilizer 0.7mol/L KCl for protoplast preparation and 17% sucrose for regeneration. The protoplast preparation rate of bacteria strains AUH-HM195 was 93.2% and regeneration rate was 14.5% under the optimal conditions. The optimal conditions of S.cerevisiae were as follows: bacterial age 16h, snailase concentration 2%, hydrolysis temperature 30℃, hydrolysis time 30min, osmotic pressure stabilizer 0.8 mol/L sorbitol, pretreated with 0.05 mol/L EDTA-Na2 and 0.2%β-ME for 20min. The protoplast preparation rate of S.cerevisiae was 85.1% and regeneration rate was 3.9% under the optimal conditions. The optimal conditions of bacteria strains R5 were as follows: bacterial age 8h, lysozyme concentration 0.5mg/mL, enzymolysis time 30min, osmotic pressure stabilizer 0.7mol/L KCl for protoplast preparation and 17% sucrose for regeneration. Both S.cerevisiae and R5 were observed to fuse with strain AUH-HM195 under microscope. However, such fused cells capable of growing and biotransforming daidzein in to O-Dma under aerobic conditions were not found.