细胞膜磷脂棕榈酸含量对自絮凝颗粒酵母耐酒精能力的影响及作用机制

研究揭示细胞膜磷脂脂肪酸组成与酵母菌耐酒精能力的一种新颖关系及其机制。分别培养于添加 0 6mmol L棕榈酸、亚油酸或亚麻酸不同条件下的自絮凝颗粒酵母 ,其细胞膜富含各自所添加的脂肪酸。细胞膜富含棕榈酸、亚油酸或亚麻酸的三种菌体于 30℃经 2 0 %(v v)酒精冲击 6h的存活率分别为 5 2 %、1 8%和 0。通过考察三种菌体于 30℃在 1 5 %(v v)酒精冲击下的细胞膜透性发现 ,细胞膜富含棕榈酸的菌体的胞外核苷酸平衡浓度分别仅为细胞膜富含亚油酸或亚麻酸菌体的 48%和 32 %,其细胞膜透性系数 (P′)分别仅为后者的 37%和 2 0 %,且三者的胞外核苷酸浓度和P′由小到大的排列顺序均与它们的存活率由高到低的排列顺序完全一致。因此 ,细胞膜富含棕榈酸的菌体具有较强的耐酒精能力是与其在高浓度酒精冲击下可维持较低的细胞膜透性密切相关的 的。     

细胞膜蛋白质氨基酸组成对自絮凝颗粒酵母耐酒精能力的影响及作用机制

揭示细胞膜组分与酵母菌耐酒精能力的一种新颖关系及其机制。实验显示 ,培养于添加和未添加 3种氨基酸 (异亮氨酸、甲硫氨酸和苯丙氨酸 ,添加浓度分别为 1 0、0 5和 2 0g L)条件下的自絮凝颗粒酵母于 30℃经2 0 % (V V)酒精冲击 9h的存活率分别为 5 7%和 0 ,表明添加该 3种氨基酸能显著提高菌体的耐酒精能力。细胞膜蛋白质氨基酸组成分析和细胞膜流动性测定表明 ,所添加 3种氨基酸是通过组入菌体细胞膜、改变细胞膜流动性从而提高菌体的耐酒精能力的 ,即当细胞膜蛋白质氨基酸组成中异亮氨酸、甲硫氨酸和苯丙氨酸含量明显增加时 ,菌体能有效抵抗高浓度酒精冲击引发的细胞膜流动性的升高 ,从而维持细胞膜的稳定。细胞膜蛋白质氨基酸组成会影响细胞膜的流动性 (膜蛋白中异亮氨酸、甲硫氨酸和苯丙氨酸含量明显增加时膜流动性降低 )是一种新的实验现象。     

膜蛋白氨基酸组成通过改变膜流动性影响粟酒裂殖酵母和酿酒酵母融合株耐酒精能力

实验显示,一种氨基酸混合液（含异亮氨酸、甲硫氨酸和苯丙氨酸,添加浓度分别为1.0、0.5和2.0g/L）能显著提高自絮凝酵母——粟酒裂殖酵母和酿酒酵母融合株SPSC的耐酒精能力。实验将菌体分别培养于添加（试验组）和未添加（对照组）该氨基酸混合液的条件下,然后收集菌体进行酒精（20%,V/V）冲击试验（30℃,9h）,结果,试验组的菌体尚有一半以上的存活细胞,而对照组的菌体全部死亡。通过对试验组和对照组的菌体细胞膜蛋白质氨基酸组成分析发现,试验组的菌体耐酒精能力提高与所添加氨基酸组入菌体的细胞膜密切相关。以DPH为荧光探针的细胞膜流动性测定分析进一步揭示,氨基酸组入菌体的细胞膜后,细胞膜能有效抵抗高浓度酒精冲击诱发的膜流动性的提高,从而维持膜的稳定。因此,实验首次揭示膜蛋白氨基酸组成可通过改变膜流动性而影响酵母菌的耐酒精能力。     

外源脂肪酸对酒精诱导自絮凝颗粒酵母细胞膜磷脂脂肪酸组成变化的影响

实验将自絮凝颗粒酵母培养于同时添加脂肪酸 (0.6mmol/L)和酒精 (6 %～ 9% ,V/V)条件下以考察其细胞膜磷脂脂肪酸组成的变化。与单独添加棕榈酸相比 ,同时添加酒精引起细胞膜磷脂棕榈酸含量明显增加 ,伴随 9十四碳烯酸、棕榈油酸和油酸含量明显减少 ;与单独添加亚油酸相比 ,同时添加酒精未引起细胞膜磷脂亚油酸含量明显变化 ,但引起油酸含量明显增加 ,伴随 9 十四碳烯酸、棕榈油酸和棕榈酸含量减少 ;与单独添加亚麻酸相比 ,同时添加酒精引起细胞膜磷脂亚麻酸含量减少 ,伴随油酸含量显著增加 ,同时 9 十四碳烯酸、棕榈油酸和棕榈酸含量减少。存活率实验证实 ,上述变化是菌体对酒精刺激的适应性响应 ,因为 ,与培养于仅添加脂肪酸条件下的菌体相比 ,培养于同时添加酒精条件下的菌体耐酒精能力明显提高。研究表明 ,棕榈酸和油酸都可通过加强细胞膜渗透屏障而提高菌体的耐酒精能力 ,这是饱和脂肪酸 (SFA)与不饱和脂肪酸 (UFA)可提高同一菌株耐酒精能力的新的实验现象 ,揭示UFA与SFA在影响酵母菌耐酒精能力的机制上存在共同的作用方式     

酿酒酵母X330高浓度发酵时耐酒精性能的初步研究

在完全合成培养基条件下,就渗透压保护剂和营养物质对一株产高浓度酒精的酿酒酵母X330高浓度发酵时耐酒精性能的影响进行了初步研究。结果表明,与渗透压相比,营养缺乏对酿酒酵母高浓度发酵时酒精耐受性能可能起着更为关键和重要的作用。发酵培养基中各营养元素对耐酒精性能的影响不同,由高到低的顺序是酵母抽提物>蛋白胨>硫酸镁>维生素C=磷酸二氢钾>氯化钙=硫酸铵。渗透压保护剂(甘氨酸和脯氨酸)能有效提高菌体酒精耐受性能。当甘氨酸添加浓度为20mmol/L或脯氨酸添加浓度为10mmol/L时,发酵终点酒精浓度最高,菌体于30℃在18%(V/V)酒精冲击下的存活率最大,且均高于对照组(未添加甘氨酸且未添加脯氨酸)水平,但甘氨酸的促进作用强于脯氨酸。     

絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制

首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。     

细胞膜磷脂脂肪酸组成对自絮凝酵母质膜ATP酶响应酒精刺激的影响及其与菌体耐酒精的关系

研究揭示细胞膜磷脂脂肪酸组成与质膜ATP酶在酵母菌耐酒精中的一种新颖关系。实验表明,细胞膜磷脂脂肪酸组成特点对生长于未添加酒精条件下的自絮凝颗粒酵母质膜ATP酶活性没有影响,但却明显影响生长于添加酒精(1％～10％,V／V)条件下的菌体质膜ATP酶对酒精激活的敏感性：预培养于添加0．6mmol／L棕榈酸、亚油酸、或亚麻酸条件下的菌体的质膜ATP酶的最大激活水平分别为各自酶的基态水平(未激活)的3．6、1．5和1．2倍,而对照组(预培养于未添加脂肪酸条件下的菌体)的相应值为2．3倍,酶产生上述最大激活水平时的酒精浓度分别为7％、6％、6％、和7％(V/V)。酶激活后米氏常数Km、最适pH和对钒酸钠(质膜ATP酶特异性抑制剂)的敏感性等性质不变,但最大反应速度υmax明显增加。实验表明,细胞膜磷脂脂肪酸组成特点对提高菌体的耐酒精能力越有利,则其质膜ATP酶被酒精激活的幅度越大,说明菌体耐酒精能力的提高与其质膜ATP酶对酒精激活的敏感性的增加密切相关。细胞膜磷脂脂肪酸组成会影响酵母菌质膜ATP酶对酒精激活的敏感性是观察到的新的实验现象。     

细胞膜磷脂脂肪酸组成对自絮凝酵母质膜ATP酶响应酒精刺激的影响及其与菌体耐酒精的关系

研究揭示细胞膜磷脂脂肪酸组成与质膜ATP酶在酵母菌耐酒精中的一种新颖关系。实验表明 ,细胞膜磷脂脂肪酸组成特点对生长于未添加酒精条件下的自絮凝颗粒酵母质膜ATP酶活性没有影响 ,但却明显影响生长于添加酒精 (1 %～ 10 % ,V/V)条件下的菌体质膜ATP酶对酒精激活的敏感性 :预培养于添加 0.6mmol L棕榈酸、亚油酸、或亚麻酸条件下的菌体的质膜ATP酶的最大激活水平分别为各自酶的基态水平 (未激活 )的 3.6、1.5和 1.2倍 ,而对照组 (预培养于未添加脂肪酸条件下的菌体 )的相应值为2.3倍 ,酶产生上述最大激活水平时的酒精浓度分别为 7%、6 %、6 %、和 7% (V/V)。酶激活后米氏常数K_m 、最适pH和对钒酸钠 (质膜ATP酶特异性抑制剂 )的敏感性等性质不变 ,但最大反应速度v_max明显增加。实验表明 ,细胞膜磷脂脂肪酸组成特点对提高菌体的耐酒精能力越有利 ,则其质膜ATP酶被酒精激活的幅度越大 ,说明菌体耐酒精能力的提高与其质膜ATP酶对酒精激活的敏感性的增加密切相关。细胞膜磷脂脂肪酸组成会影响酵母菌质膜ATP酶对酒精激活的敏感性是观察到的新的实验现象.     

Ca2+对丹参培养细胞中迷迭香酸合成及其相关酶活性的影响

探究了外界Ca2+(0~50 mmol/L)对丹参培养细胞迷迭香酸合成及其相关酶活性的影响,并利用细胞膜钙离子通道抑制剂异搏定(Verpamil,VP)及钙离子载体A23187初步探讨了外界Ca2+浓度变化影响丹参培养细胞次生代谢的机制。结果显示:培养6 d时的丹参细胞中迷迭香酸积累量与外界Ca2+浓度显著相关,其中10 mmol/L Ca2+最有利于迷迭香酸的合成,迷迭香酸最大积累量达20.149 mg/g DW,比1 mmol/L和3 mmol/LCa2+处理分别高37.3%和20.4%。分析迷迭香酸合成的两条支路上的关键酶PAL和TAT活性变化发现,两种酶活性亦受外界Ca2+浓度影响,且活性变化先于迷迭香酸的积累,说明这两种酶均参与迷迭香酸的生物合成,但PAL比TAT促进作用更明显。进一步用VP和A23187处理发现,外界Ca2+影响迷迭香酸的合成是通过影响胞内Ca2+浓度实现的,胞外Ca2+内流可能参与了这一过程。     

絮凝颗粒粒度分布对自絮凝酵母SPSC01乙醇耐受能力的影响

利用激光聚焦反射式颗粒测量系统, 通过调节不同的搅拌速率, 得到了分批补料培养条件下粒度分布不同的四个絮凝酵母SPSC01颗粒群体, 进而对絮凝颗粒群体分布对乙醇耐受性进行了系统研究。经过6 h、20%乙醇的冲击, 颗粒粒度为100、200、300和400 mm的自絮凝酵母SPSC01的存活率分别为3.5%、26.7%、48.8%和37.6%。这表明不同粒度分布的絮凝颗粒群体乙醇耐受性具有明显差别, 在一定粒度范围内乙醇耐受性达到最高, 乙醇耐受性最高的酵母群体的乙醇得率系数85.5%, 比乙醇耐性最低的颗粒群体提高了7.2%。粒度为100、200和300 mm的自絮凝酵母颗粒群体总麦角固醇、游离麦角固醇及海藻糖含量与粒度大小成正相关, 但在粒度为400 mm的絮凝颗粒群体中总麦角固醇、游离麦角固醇及海藻糖含量呈下降趋势, 与其乙醇耐性低于300 mm絮凝颗粒的结果相一致。对细胞膜透性的研究表明, 颗粒粒度为300 mm的絮凝酵母颗粒细胞膜通透性(P′)最低, 分别仅为颗粒粒度为100 mm和200 mm颗粒群体的43%和52%, 表明粒度分布不同的絮凝颗粒群体乙醇耐性的差别与细胞膜透性密切相关。     

Enhancing ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae by Mg2+ via reduction in plasma membrane permeability

Mg²⁺ at 3.5 mM increased the tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae to ethanol. After 9 h of exposure to 20% (v/v) ethanol at 30 °C, all cells died whereas over 50% remained viable for the cells grown with Mg²⁺. The effect of Mg²⁺ is closely related to its ability to decrease plasma membrane permeability of cells subjected to ethanol stress.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae.

Ethanol, in concentrations that affect growth and fermentation rates (3 to 10% [vol/vol]), activated in vivo the plasma membrane ATPase of Saccharomyces cerevisiae. The maximal value for this activated enzyme in cells grown with 6 to 8% (vol/vol) ethanol was three times higher than the basal level (in cells grown in the absence of ethanol). The Km values for ATP, the pH profiles, and the sensitivities to orthovanadate of the activated and the basal plasma membrane ATPases were virtually identical. A near-equivalent activation was also observed when cells grown in the absence of ethanol were incubated for 15 min in the growth medium with ethanol. The activated state was preserved after the extraction from the cells of the membrane fraction, and cycloheximide appeared to prevent this in vivo activation. After ethanol removal, the rapid in vivo reversion of ATPase activation was observed. While inducing the in vivo activation of plasma membrane ATPase, concentrations of ethanol equal to and greater than 3% (vol/vol) also inhibited this enzyme in vitro. The possible role of the in vivo activation of the plasma membrane proton-pumping ATPase in the development of ethanol tolerance by this fermenting yeast was discussed.     

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity: role of intracellular calcium

The mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity to isolated hepatocytes was studied. MPTP was more toxic to hepatocytes than its major metabolite, 1-methyl-4-phenylpyridine (MPP+); this may, in part, be explained by the lesser permeability of the hepatocyte plasma membrane to the cation compared to its parent compound, MPTP. Loss of cell viability was preceded by plasma membrane bleb formation and disturbance of intracellular Ca2+ homeostasis. MPTP caused a rapid depletion of the mitochondrial Ca2+ pool which was followed by a marked and sustained elevation of cytosolic free Ca2+ concentration. This increase of cytosolic Ca2+ level appeared to be associated with the impairment of the cell's Ca2+ extrusion system since the plasma membrane Ca2+-ATPase was markedly inhibited in MPTP-treated hepatocytes. Preincubation of hepatocytes with inhibitors of monoamine oxidase type B, but not A, protected the cells from MPTP-induced cytotoxicity. Moreover, the monoamine oxidase B inhibitor, pargyline, prevented the rise in cytosolic free Ca2+ concentration and partially protected the plasma membrane Ca2+-ATPase from inhibition by MPTP. As observed with MPTP, MPP+ caused an extensive loss of mitochondrial Ca2+ and significantly decreased the rate of Ca2+ efflux from hepatocytes. However, MPP+ was without effect on the plasma membrane Ca2+-ATPase. In conclusion, our studies demonstrate that MPTP caused a substantial elevation of cytosolic Ca2+ which preceded loss of cell viability and we propose that calcium ions are of major importance in the mechanism of MPTP- and MPP+-induced toxicity in hepatocytes.     

Ethanol tolerance and the variation of plasma membrane composition of yeast floc populations with different size distribution

The ethanol tolerance of a self-flocculating yeast strain SPSC01 was investigated in an oxygen-limited fed-batch bioreactor. Employing Focused Beam Reflectance Measurement (FBRM) on-line monitoring system, four yeast floc populations with the average size ranging from 100 to 400mum were obtained. It was found that ethanol tolerance increased with the increasing floc size in the 100, 200, and 300mum floc populations, while increasing the average floc size further to 400mum resulted in lower ethanol tolerance. Examination of the membrane composition of different floc populations revealed that the plasma membrane composition of the floc populations was significantly different in the contents of ergosterol, phosphatidylinositol, as well as phospholipid palmitoleic acid. What's more, the plasma membrane of more ethanol tolerant floc population was less permeable when subjected to 15% (v/v) ethanol shock treatment, and the plasma membrane ATPase activities were higher in the floc populations with higher ethanol tolerance. These results indicate that the average size distribution of the floc populations exerted great influence on the physiological status of yeast cells during the ethanol production process, leading to the changes in plasma membrane composition that contributed to improved ethanol tolerance in self-flocculating yeast SPSC01.