大豆异黄酮转化菌株原生质体制备与融合研究
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摘要
大豆异黄酮是大豆在其生长过程中形成的次级代谢产物,主要由黄豆苷原、染料木素和黄豆黄素组成。它被机体吸收以后能与雌激素受体结合发挥类雌激素效应,具有防治癌症、降低血脂、抗氧化、预防骨质疏松和改善妇女更年期综合症等多种生理功能。大豆异黄酮在胃肠道微生物菌群作用下可被转化为各种不同代谢产物,如二氢黄豆苷原(DHD)、雌马酚(Equol)和去氧甲基安哥拉紫檀素(O-Dma)等。最新研究表明大豆异黄酮代谢产物具有比大豆异黄酮更高更广的生物学活性。
原生质体融合技术是用物理或化学方法诱导遗传特性不同的两亲本原生质体融合为一个新细胞,使两亲株的整套基因相互重新组合,最后获得稳定融合子的技术。由于去除了细胞壁,原生质体膜易于融合,更容易发生基因组的融合重组。
本实验室分离的AUH-HM195(Enterococcus hirae)菌株是一株兼性厌氧细菌,在有氧和厌氧条件下均可生长,但只有在严格厌氧条件下可以将黄豆苷原转化为去氧甲基安哥拉紫檀素。为了使转化菌株在有氧条件下也具有转化功能,本试验尝试将大豆异黄酮转化菌株与好氧菌进行原生质体的融合。供试好氧菌包括酿酒酵母(Saccharomyces.cerevisiae)和本实验室分离的专性好养细菌Pseudomonas aeruginosa R5。AUH-HM195菌株原生质体制备与再生的最优组合为:菌龄6h、溶菌酶浓度5mg/mL、酶解时间1h、渗透压稳定剂在制备时使用0.7mol/L KCl、再生时为17%蔗糖,在此条件下制备率可达93.2%,再生率14.5%。酿酒酵母在菌龄16h,以2.0%蜗牛酶进行酶解,酶解温度30℃,酶解时间30min,0.8mol/L山梨醇做渗透压稳定剂,以0.05mol/L EDTA-Na2溶液和0.2 %β-巯基乙醇为预处理剂处理20min时为最佳,在此优化条件下,原生质体形成率为85.1%,再生率为3.9%;R5在菌龄8h,酶浓度为0.5mg/mL,酶解时间30min,制备时使用0.7mol/L KCl,再生时以17%蔗糖为渗透压稳定剂时R5菌株可以达到最高的制备率和再生率。在显微镜下观察到了酿酒酵母和R5菌株均可与AUH-HM195菌株原生质体融合,但最终未能筛选到能在有氧条件下生长并具转化功能的融合子。
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.
原生质体融合技术是用物理或化学方法诱导遗传特性不同的两亲本原生质体融合为一个新细胞,使两亲株的整套基因相互重新组合,最后获得稳定融合子的技术。由于去除了细胞壁,原生质体膜易于融合,更容易发生基因组的融合重组。
本实验室分离的AUH-HM195(Enterococcus hirae)菌株是一株兼性厌氧细菌,在有氧和厌氧条件下均可生长,但只有在严格厌氧条件下可以将黄豆苷原转化为去氧甲基安哥拉紫檀素。为了使转化菌株在有氧条件下也具有转化功能,本试验尝试将大豆异黄酮转化菌株与好氧菌进行原生质体的融合。供试好氧菌包括酿酒酵母(Saccharomyces.cerevisiae)和本实验室分离的专性好养细菌Pseudomonas aeruginosa R5。AUH-HM195菌株原生质体制备与再生的最优组合为:菌龄6h、溶菌酶浓度5mg/mL、酶解时间1h、渗透压稳定剂在制备时使用0.7mol/L KCl、再生时为17%蔗糖,在此条件下制备率可达93.2%,再生率14.5%。酿酒酵母在菌龄16h,以2.0%蜗牛酶进行酶解,酶解温度30℃,酶解时间30min,0.8mol/L山梨醇做渗透压稳定剂,以0.05mol/L EDTA-Na2溶液和0.2 %β-巯基乙醇为预处理剂处理20min时为最佳,在此优化条件下,原生质体形成率为85.1%,再生率为3.9%;R5在菌龄8h,酶浓度为0.5mg/mL,酶解时间30min,制备时使用0.7mol/L KCl,再生时以17%蔗糖为渗透压稳定剂时R5菌株可以达到最高的制备率和再生率。在显微镜下观察到了酿酒酵母和R5菌株均可与AUH-HM195菌株原生质体融合,但最终未能筛选到能在有氧条件下生长并具转化功能的融合子。
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.
引文
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