【作者】 陈慧黠；

【导师】 修志龙； 白凤武；

【作者基本信息】 大连理工大学 ， 生物化工， 2010， 博士

【摘要】 大气压介质阻挡放电(DBD)等离子体中含有大量的电子、离子、激发态原子和分子及自由基等活性粒子,以及电场、紫外线,能在较短时间内达到很高的灭菌效率,并不损害灭菌物质表面结构,同时具备低温、易操作等优点,受到越来越多的关注。然而DBD等离子体对微生物的作用机理还不是很清楚,将其用于诱变微生物的相关研究工作开展较少,尤其是诱变真菌尚未见报道。本论文着重研究了大气压DBD空气等离子体对酿酒酵母(Saccharomyces cerevisiae)的生物学效应以及对产乙醇菌株S. cerevisiae ATCC 4126和Candida shehatae CICC1766的诱变效应,主要研究结果如下：1.等离子体处理对酵母细胞具有显著的生物学效应。大气压DBD空气等离子体在均方根(root-mean-square, RMS)放电电压为12.0 kV,频率为20 kHz,放电间隙为4 mm,处理S. cerevisiae菌液深度为2 mm的条件下,引起酵母细胞在短时间内大量死亡。通过吉姆萨染色发现等离子体处理时间越长,细胞着色越深；S. cerevisiae胞外蛋白质和核酸含量随着处理时间的延长显著增加,表明等离子体可能破坏了酵母细胞的细胞膜,引起细胞通透性增加,导致胞内蛋白质和核酸渗漏到胞外。同时,等离子体处理引起细胞生长延迟,细胞周期阻滞于G1期,表明DNA可能受到损伤。2.等离子体诱导酵母细胞的氧化应激。等离子体处理不仅能使去离子水中的活性氧化物含量显著增加,而且也使S. cerevisiae胞内的活性氧物质(reactive oxygen species, ROS)含量显著增加,酵母胞内、外总抗氧化能力(total antioxidant capability, T-AOC)和胞内谷胱甘肽还原酶(glutathione reductase, GR)活力都有不同程度提高,丙二醛含量也随着处理时间的延长而不断增加。等离子体处理后再将细胞培养3h,胞内超氧化物歧化酶(superoxide dismutases, SOD)和过氧化氢酶(catalase, CAT)比活力依然增强。这些结果表明,DBD空气等离子体对酵母菌产生了一定的氧化损伤,并引发酵母细胞的氧化应激。除了直接作用于细胞表面导致细胞损伤甚至死亡外,等离子体引起的细胞内氧化应激也可能是引起细胞损伤、甚至死亡的重要原因之一。3.等离子体对酿酒酵母产乙醇能力的刺激效应。用DBD空气等离子体处理S.cerevisiae菌,筛选耐高温、耐高浓度底物的高产乙醇菌株。将等离子体处理4 min筛选的单菌落接种到葡萄糖浓度为30g/L的培养基中,在40℃下培养,获得8株具有正效应的菌株,其乙醇产量较对照菌株提高13.7～40.4%。正效应菌株在葡萄糖浓度为100 g/L的培养基中370C下培养作进一步的验证,其中的5株菌仍保持良好的乙醇生产性能,乙醇产量分别比对照提高了2.5～6.6%；提高培养温度和底物葡萄糖的初始浓度作进一步的筛选,在葡萄糖初始浓度分别为100 g/L和300 g/L的培养基中40℃培养,发现先前的几株菌未显示出高产乙醇的优势,而重新诱变筛选出的菌株则表现出高产特性,但经多次传代培养后,高产性状丧失。这些结果表明等离子体对S. cerevisiae菌体有一定的激活作用,可以获得耐高温、耐高浓度底物并高产乙醇的正效应菌株,但获得稳定遗传的正突变菌株还有待进一步研究。4.等离子体对利用木糖产乙醇的休哈塔假丝酵母细胞的诱变效应。用DBD空气等离子体处理C. shehatae CICC1766,诱变、筛选高效利用木糖生产乙醇的菌株。经过TTC指示剂法平板筛选和摇瓶发酵实验的验证,继代培养15次获得3株性状变化明显且稳定遗传的突变株,分别命名为C80828, C81015和C81020。其中C80828, C81015为正突变株,在TTC平板上显色比野生菌株深,在木糖培养基中的乙醇产量也显著高于野生型菌株。在50g/L木糖发酵培养基中,到发酵至120 h时,C80828的乙醇产量比对照高出8.2%；C81015的产量高出36.2%(P<0.05)。但C81015的生物量明显低于对照,表明C81015细胞的比乙醇生成速率显著提高。研究发现C81015中NADH/NADPH-木糖还原酶(xylose reductase, XR)和NAD+-木糖醇脱氢酶(xylitol dehydrogenase, XDH)比活力分别比对照提高了34.1%(P<0.01),61.5%(P<0.05)和66.3%(P<0.01)。而以葡萄糖为底物时,C81015的乙醇产量几乎与野生菌株相同,表明DBD空气等离子体影响C. shehatae木糖代谢关键酶XR和XDH的活力,从而影响C. shehatae突变株利用木糖产乙醇的能力。而菌株C81020为负突变株,与野生菌株相比,以木糖为底物产乙醇的量很低,两个关键酶活力也显著降低,但生物量有所提高。SDS-PAGE分析表明其胞内总蛋白图谱与野生型明显不同,表明等离子体可能对该菌株产生了遗传毒性。5.等离子体对木糖利用关键酶的突变效应。对突变株C81015和野生菌株木糖还原酶和木糖醇脱氢酶的基因XYL1和XYL2的序列进行检测和比对。序列比对结果发现,与野生型菌株相比,突变株C81015 XYL1中有3个碱基不同,从野生型菌株到突变株C81015的碱基改变(ATT→GGT, AAT→AAG)导致了氨基酸残基的变化(Ile309→Gly309, Asn314→Lys314)。而在XYL2序列对比中发现,突变株C81015与野生型有6处碱基不同,分别位于序列的前、中、后部。其中基因序列中部碱基的不同(AGT->GGT, TTC→CTC)导致了氨基酸残基变化(Ser185→Gly185, Phe189→Leu189)。而位于基因序列前、后部的不同碱基并未引起相应位置氨基酸残基的改变。总之,DBD空气等离子体产生的ROS是导致酵母损伤以及死亡的重要原因之一。DBD空气等离子体能引起酵母细胞的氧化应激,导致胞内DNA和蛋白质损伤、基因的改变。因此DBD空气等离子体除了应用于物质的表面灭菌外,还能用来诱变S. cerevisiae和木糖利用菌株C. shehatae,来筛选高产乙醇的突变株。更多还原

【Abstract】 Atmospheric pressure Dielectric Barrier Discharge (DBD) air plasma composes of electron, ions, excited atoms and molecules, free radicals, and other active particles, as well as electric field and ultraviolet radiation, and thus is an efficient way for non-invasive surface sterilization. In addition, the plasma with low temperature is easy to be operated. Thus, it has attracted more and more attention. However, the mechanism of the plasma on micro-organisms sterilization is still not clear. On the other hand, the plasma, as a source of various active particles, has rarely been used as a mutagen for strain development. In this study, the biological effect of the plasma on the yeast Saccharomyces cerevisiae were investigated, and its mutagesis on ethanologenic strains, S. cerevisiae ATCC 4126 and Candida shehatae CICC1766 were further examined.1. The biological effect induced by the plasma on S. cerevisiae. The plasma was obtained at a root-mean-square (RMS) voltage of 12.0 kV and a frequency of 20 kHz. S. cerevisiae cells were suspended in water with a pool of 2 mm in depth. A discharge gap of 4 mm between the surface of the sample and the tip of the upper electrode was applied. After plasma discharge, the cells showed extensive death. The longer the treatment time, the more intense stained color the cells exhibited. And in the meantime, the plasma-treated cells exhibited significant increases in extracellular protein and nucleic acid concentrations, suggesting that these macromolecules were released from the cells, possibly via the leakages in the cell membranes. In addition, retardance in cell growth also occurred, and the plasma-treated cells showed cell cycle arrested at their G1 phase, and this arrest effect increased with the increase of the treatment time, indicating the presence of intracellular DNA damage.2. Oxidative stress induced by the plasma on S. cerevisiae. For the plasma-treated S. cerevisiae cells, the concentration of reactive oxygen species (ROS) within the cells increased significantly, leading to the activation of total intracellular and extracellular antioxidant capability (T-AOC), and intracellular glutathione reductase (GR). Intracellular malondialdehyde (MDA) content also increased with the increase of the plasma treatment time. After 3 h of re-incubation following plasma treatment, the specific activities of intracellular superoxide dismutases (SODs) and catalase increased. These results indicated that the plasma might have inflicted oxidative damage on the yeast cells and exerted oxidative stress, witch could be the cause of cell damage, or even cell death.3. The stimulation effect of the plasma on ethanol production of S. cerevisiae. The plasma was used to improve the ethanol production of S. cerevisiae isolates when they were cultured under high temperature and substrate concentration conditions. Using the medium containing 30 g/L glucose and incubated at 40℃,8 clones were isolated as positive strains, and their ethanol production was enhanced from 13.7 to 40.4%, compared to their wild type. When they were cultured with the medium containing 100 g/L glucose under 37℃,5 isolates could maintain their improved ethanol production, which was enhanced from 2.5 to 6.6%, compared to their wild type. When these isolates were subsequently cultured under 100 g/L and 300 g/L glucose at 40℃, their ethanol production couldn’t maintain. More plasma-treated clones with improved ethanol production were selected with 300 g/L glucose medium and under 40℃; however, they couldn’t maintain their improved ethanol production with subcultures, either, indicating that the plasma might have some stimulate effect on S. cerevisiae cells for a while, and further work needs to be done to obtain stable positive mutants.4. The mutation effect of the plasma on the xylose-fermenting yeast C. shehatae. The plasma was used to enhance ethanol production of the xylose-fermenting yeast, C. shehatae CICC1766. Three stable mutants, C80828, C81015 and C81020 were isolated by 15 subcultures that switched between TTC medium incubation and flask culture. Among them, C80828 and C81015, which showed more intense red color on the TTC plate, and exhibited higher ethanol production, compared to the wild type were designated as positive mutants. With the medium containing 50 g/L xylose, C80828 enhanced ethanol production by 8.2%, and C81015 by 36.2%, compared to their wild type (P<0.05). However, the biomass production of C81015 was significantly lower than that of its wild type, indicating its specific ethanol productivity of C81015 increased significantly. At the same time, the specific activities of NADH- and NADPH-linked xylose reductases (XR) and NAD+-linked xylitol dehydrogenase (XDH) of C81015 (as measured in the cell extract) increased by 34.1%(P<0.01),61.5%(P<0.05) and 66.3%(P<0.01), respectively, compared to its wild type. However, no difference in ethanol production from glucose between C81015 and its wild type was detected. In contrast, C81020 was a negative mutant and showed decrease in ethanol production, with significantly lower XR and XDH specific activities. These results indicated that the DBD air plasma might affect with XR and XDH to influent the ethanol production of mutant C81015. In addition, more biomass was harvested in xylose-containing medium of C81020. SDS-PAGE of the total intracellular proteins of C81020 showed bands that differed from its wild type, suggesting that the plasma might have genotoxic effect on the C. shehatae.5. The mutagenesis of the plasma on key enzymes for xylose consumption. The genes XYL1 and XYL2, which encodes for XR and XDH, respectively, in the mutant C81015 and the wild type were sequenced. The XYL1 sequence of C81015 had three nucleotide changes compared to its wild type, resulting in the codon changes ATT->GGT and AAT→AAG, which corresponds to two amino acid substitutions, Ile309→Gly309 and Asn314→Lys314. In the XYL2 sequence of C81015, six nucleotides were found to be different from the wild type, covering the beginning, middle and end parts of the sequence. However, only the nucleotide changes in the middle of the sequence, AGT→GGT and TTC→CTC, resulted in amino acid substitution, Ser185→Gly185 and Phe189→Leu189, respectively.In conclusion, ROS produced in the DBD air plasma was one of the most important factors causing cell damage, even cell death. The DBD air plasma can induce oxidative stress in yeast cells, cause DNA damage, protein leakage and gene changes, which can be used to generate mutants from yeast with improved ethanol production, either regular S. cerevisiae or xylose-fermenting C. shehatae in addition to sterilization.更多还原