酵母菌中超氧化物歧化酶的研究

首次提出了用甲苯破壁提取酵母菌中SOD的方法,此法提取SOD的含量分别为氯仿-乙醇法、酶裂解法和细胞自溶法的1.29、1.08和2.01倍.通过对5种酵母菌的测定,发现酵母菌对氧的抗性与其SOD含量密切相关;同时对一株SOD含量较高的卡尔酵母(Saccharomyces carlsbergensis)BY2菌株进行了营养和培养条件的研究,结果表明它们对该菌株SOD含量和SOD同功酶的影响较大.     

酵母超氧物歧化酶高产菌的选育

采用常规筛选方法从300多株不同种属的酵母菌中筛选到两株细胞生物量和超氧物歧化酶(SOD)含量都较高的菌株(酿酒酵母,编号为Y-8和Y一111)作为实验出发菌.经单倍体分离、N-甲基-N-亚硝基-N’-硝基胍(MNNG)诱变和群体杂交等手段,从中选育出一株细胞生物量略高于实验出发菌、超氧物歧化酶高达1350U/g湿菌体的SOD高产菌株(编号为ZDF-48),它的SOD产量分别为实验出发菌株Y-8和Y-111的2.2 倍和2.4倍.经分离纯化后蛋白含量及超氧物歧化酶活性测定等研究,证明我们选育出的ZDF-48是一株生产超氧物歧化酶的优良品系.     

酵母菌中SOD复合酶的初步研究

对不同酵母菌中ＳＯＤ等抗氧化酶的活性进行了初步的分析测定,筛选出了一株诸酶活性都较高的菌株（丹宝利面包活性干酵母）。研究了该酵母在不同培养时期ＳＯＤ等酶少力的变化情况,发现ＰＯＤ、ＣＡＴ等酶的活性水平ＳＯＤ活性的变化有密切的相关性。通过比较几种提取方法的效果,认为利用甲苯破壁法提取ＳＯＤ复合酶具有一定的可行性。     

利用原生质体诱变育种选育富硒能力强的酵母菌株*

利用原生质体诱变育种技术选育富硒能力强的酵母菌株,从13株啤酒酵母中筛选出一株富硒量高的诱变出发菌株,采用溶壁酶进行破壁,确定了原生质体制备的最适条件为酶浓度1g/100mL,酶解处理时间为120min,原生质体形成率为95.2%,再生率为21.8%,诱变后筛选出富硒量为821mg/kg,酵母干菌体收获量为0.88g/100mL的酵母菌A1。     

遮荫条件下黄檗生长和生理响应的性别差异研究

分析遮荫条件下黄檗幼苗形态指标、生理指标和代谢物质性别间差异，探讨其对遮荫的响应策略，为黄檗野生资源保护及造林抚育提供理论依据。以2年生黄檗雌雄株幼苗为试验材料，采用控制实验，设置3个遮荫梯度，自然光为对照，对比研究遮荫条件下黄檗幼苗雌雄植株形态特征、生物量分配、抗氧化防御酶活性、代谢物质的差异，以揭示其遮荫条件下的生存策略。结果表明：遮荫处理更有利于黄檗的生长，轻度遮荫雌株生物量积累大于雄株，重度遮荫后雄株大于雌株；遮荫处理对叶绿素a、总叶绿素含量均有显著影响（P<0.01），性别间差异不显著，遮荫条件有利于促进黄檗幼苗叶绿素积累；遮荫处理对酶活性和MDA含量均有显著影响，性别间差异不显著，SOD酶活性均随遮荫强度的增强而增大，POD和CAT酶活性随遮荫强度的增强而减小，MDA含量在对照处理时最高；遮荫条件下可溶性糖和可溶性淀粉含量下降，性别间差异不显著。黄檗幼苗通过改变构件性状、生物量、抗氧化酶活性及代谢物质含量，适应遮荫环境。研究遮荫对黄檗生长和生理响应的性别差异，以期为遮荫环境下黄檗个体发育、种群形成、适应机制及种群繁衍等研究奠定基础，为雌雄异株植物资源的保护利用提供科学借鉴。     

利用原生质体诱变育种选育富硒能力强的酵母菌株

利用原生质体诱变育种技术选育富硒能力强的酵母菌株,从13株啤酒酵母中筛选出一株富硒量高的诱变出发菌株,采用溶壁酶进行破壁,确定了原生质体制备的最适条件为酶浓度1g／100mL,酶解处理时间为120min,原生质体形成率为95．2％,再生率为21．8％,诱变后筛选出富硒量为821mg／kg,酵母干菌体收获量为0．88g／100mL的酵母菌Al。     

酵母SOD形成的生理学研究

对筛选出的一株ＳＯＤ高产菌株Ｙ－２１６形成ＳＯＤ的生理条件作了初步研究。结果表明：碳源、氮源、碳氮配比和金属离子等营养条件及培养温度、时间和通气量等培养条件,均对该菌株生物量与ＳＯＤ含量有较大的影响。同时通过对几种酵母的测定,发现酵母菌对氧的抗性越大其ＳＯＤ含量也就越高。     

产角质酶酵母菌的发现与鉴定及产酶条件的优化

为得到有效水解杜仲角质层的角质酶,通过生物酶法提取杜仲中的活性成分。从病变的杜仲叶中分离出一株能高产角质酶的菌种,鉴定分析证实该菌株是胶红酵母(Rhodotorula mucilaginosa),这是首次从植物病原真菌以外发现产角质酶的酵母菌,并对该菌株发酵产角质酶的条件进行优化。这一发现意义重大,因为酵母菌比植物病原菌安全许多,更利于大规模生产。     

土壤水分对异株荨麻（Urtica dioica）保护酶和渗透调节物质的影响及其与叶片光合和生物量的相关性

通过对不同土壤水分处理下异株荨麻保护酶系(SOD、POD)活性、渗透调节物质(可溶性蛋白、可溶性糖和游离脯氨酸)和细胞膜脂过氧化产物(MDA)含量以及最大光合速率、生物量的测定,各项生理指标间线性相关性的分析,探讨异株荨麻对水分变化的响应特性和耐干旱适应能力.实验采用盆栽水分梯度法将异株荨麻扦插苗按土壤相对含水量93.58%、80.74%、67.90%、55.06%和42.22%分组,以充足水分93.58%为CK对照组,实验组水分由高到低为T1、T2、T3和T4共5组.结果表明,(1)异株荨麻体内SOD活性随水分含量下降而呈先上升后下降的变化趋势,POD活性与之成相似互补的变化趋势;可溶性蛋白、可溶性糖、游离脯氨酸和MDA含量随水分含量的下降而升高; (2)各保护酶系和渗透调节物质与土壤水分呈显著线性负相关,保护酶系、渗透调节物质与过氧化产物MDA线性正相关;(3)比较光合速率、生物量和各种酶系、渗透调节物质的关系发现,光合速率与各生理指标无明显相关性,渗透调节物质对生物量积累有显著负相关性.     

遮阴对南山茶与红皮糙果茶幼苗生长及生理特性的影响

以2年生南山茶Camellia semiserrata、红皮糙果茶C. crapnelliana实生苗为试材，研究全光照(CK)、30%遮阴、50%遮阴和70%遮阴等处理对其生长、生物量分配及叶片部分生理生化指标的影响。结果表明，30%遮阴可显著增加南山茶幼苗株高净生长量、总生物量，其叶绿素a、叶绿素b显著增加，SOD、CAT酶活性及Pro含量均与CK组无显著差异，MDA含量则显著降低；红皮糙果茶幼苗则在30%遮阴下显著增加株高和地径，叶绿素a、叶绿素b含量及SOD酶活性、Pro含量与CK组无差异，MDA含量显著下降。说明两种山茶属植物幼苗较适宜的光照条件为遮阴30%。此外，红皮糙果茶幼苗在50%遮阴条件下与CK组比较，虽然地径生长受抑制，但株高生长无显著差异，且叶绿素a、叶绿素b含量与CAT酶活性显著增加，Pro、MDA含量显著下降，表明红皮糙果茶幼苗能通过调节叶绿素含量、CAT酶活性和Pro含量积极应对50%遮阴的弱光环境，进行适应性生长。因此生产上及绿化应用中，南山茶幼苗对光照的需求更为严格，遮阴控制在30%；红皮糙果茶幼苗则可适应更加荫蔽的环境(50%遮阴)。     

Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae

The diversity and content of available nitrogen sources in the growth medium both are very important in the accumulation of ergosterol in the yeast cell membrane. Growth on the good nitrogen sources such as ammonia can harvest more yeast cells than on poor ones, but ergosterol content in those yeast cells is relatively lower. Ergosterol content, one of the most variable parameters in ergosterol production by yeast cultivation, is greatly influenced by nitrogen limitation. The aim of our work was to study how the nitrogen sources affected the membrane ergosterol content and increase the total ergosterol yield. On the premise of keeping high ergosterol content in yeast cell, the ergosterol yield was enhanced by increasing the yeast biomass. Direct feed back control of glucose using an on-line ethanol concentration monitor was introduced to achieve high cell density. Ammonia, which acted as nitrogen source, was added to adjust pH during fermentation process, but its addition needed careful control. Cultivation in 5 L bioreactor was carried out under following conditions: culture temperature 30+/-1 degrees C, pH 5.5+/-0.1, agitation speed 600 rpm, controlling ethanol concentration below 1% and controlling ammonium ion concentration below 0.1 mol/L. Under these conditions the yeast dry weight reached 95.0+/-2.6 g/L and the ergosterol yield reached 1981+/-34 mg/L.     

Continuous culture of Candida utilis: influence of medium nitrogen concentration

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h^-1), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.     

Expression of a familial amyotrophic lateral sclerosis-associated mutant human superoxide dismutase in yeast leads to decreased mitochondrial electron transport

Strains of Saccharomyces cerevisiae that express either the wild type or the amyotrophic lateral sclerosis-associated mutant human copper-zinc superoxide dismutase (SOD1) proteins A4V and G93A, respectively, in a yeast SOD1-deficient parent strain were used to investigate the hypothesis that expression of a mutant SOD1 protein causes deficient mitochondrial electron transport as a possible mechanism for disease induction. Mitochondria isolated from the wild type SOD1-expressing yeast were identical to mitochondria from the parent strain in heme content and activities of complexes II, III, and IV. Mitochondria isolated from the A4V-expressing yeast had decreased rates of electron transport in complexes II+III, III, and IV and corresponding decreases in hemes b, c-c1, and a-a3 content compared to mitochondria from wild type human SOD1-expressing yeast. Mitochondria isolated from G93A-expressing yeast had decreased rates of electron transport in complex IV and probably in complex II with a corresponding decrease in heme a-a3 content. These results suggest that mutant SOD1-expression causes defective electron transport complex assembly and that the yeast system will provide an excellent model for the study of the mechanism of mutant SOD1-induced mitochondrial electron transport defects.     

Role of superoxide dismutase in the oxidation of N-alkanes by yeasts.

Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.     

Ethanol and value‐added byproducts from rice straw by dimorphic fungus Mucor hiemalis

Different morphologies of Mucor hiemalis were induced and used for the production of ethanol and biomass from rice straw through a separate hydrolysis and fermentation process. The yield of enzymatic hydrolysis was improved from 40.4% for the untreated straw to 80–93% by employing sodium hydroxide and concentrated phosphoric acid pretreatments with or without ultrasonication. The best hydrolysis performance was achieved after pretreatment by sodium hydroxide assisted with ultrasonication. The ethanol yields from the hydrolysates were 0.39–0.44 g/g depending on the pretreatment method and the fungus morphology. The yeast‐like form of the fungus showed faster glucose assimilation and slightly higher ethanol yield compared to the other morphologies. The biomass yield of mostly yeast‐like cells was more than the other morphologies (0.202–0.282 g/g glucose). Moreover, the biomass of the yeast‐like cells had more protein content (46.7–52.4 %) compared to filamentous cells (37.7–46.3 %). The cell wall, alkali‐insoluble material (AIM) of the biomass, represented 16.3–20.1% of the biomass. On average, total chitin‐chitosan content of AIM of the biomass of purely filamentous, mostly filamentous, mostly yeast‐like, and purely yeast‐like forms of the fungus was 0.460, 0.373, 0.330, and 0.336 g/g AIM of the biomass, respectively.     

Biotransformation of benzaldehyde by Saccharomyces cerevisiae: characterization of the fermentation and toxicity effects of substrates and products

Although higher initial rates of phenylacetyl carbinol formation were observed in fermentations containing a high starting benzaldehyde level, a massive reduction in yeast viability was observed resulting in early cessation of production formation. Pulse feeding to maintain lower benzaldehyde concentrations resulted in a lower initial reaction rate, but prolonged yeast viability and the biotransformation. This resulted in higher overall product tilers. As benzaldehyde concentration was increased, yeast growth rate was reduced (0.5 g/L), inhibited (1-2 g/L), or cell viability reduced (3 g/L). Benzaldehyde appeared to alter the cell permeability barrier to substrates and products. Reductions in yeast biomass levels and especially protein and lipid content were observed during the biotransformation. The effects of benzaldehyde and reaction products on yeast pyruvate decarboxylase and alcohol dehydrogenase stability were determined. Homogenized yeast cells produced similar phenylacetyl carbinol levels to whole yeast only if supplemented with thiamine pyrophosphate and magnesium.     

Growth model and metabolic activity of brewing yeast biofilm on the surface of spent grains: a biocatalyst for continuous beer fermentation

In the continuous systems, such as continuous beer fermentation, immobilized cells are kept inside the bioreactor for long periods of time. Thus an important factor in the design and performance of the immobilized yeast reactor is immobilized cell viability and physiology. Both the decreasing specific glucose consumption rate (q(im)) and intracellular redox potential of the cells immobilized to spent grains during continuous cultivation in bubble-column reactor implied alterations in cell physiology. It was hypothesized that the changes of the physiological state of the immobilized brewing yeast were due to the aging process to which the immobilized yeast are exposed in the continuous reactor. The amount of an actively growing fraction (X(im)act) of the total immobilized biomass (X(im)) was subsequently estimated at approximately X(im)act = 0.12 g(IB) g(C)(-1) (IB = dry immobilized biomass, C = dry carrier). A mathematical model of the immobilized yeast biofilm growth on the surface of spent grain particles based on cell deposition (cell-to-carrier adhesion and cell-to-cell attachment), immobilized cell growth, and immobilized biomass detachment (cell outgrowth, biofilm abrasion) was formulated. The concept of the active fraction of immobilized biomass (X(im)act) and the maximum attainable biomass load (X(im)max) was included into the model. Since the average biofilm thickness was estimated at ca. 10 microm, the limitation of the diffusion of substrates inside the yeast biofilm could be neglected. The model successfully predicted the dynamics of the immobilized cell growth, maximum biomass load, free cell growth, and glucose consumption under constant hydrodynamic conditions in a bubble-column reactor. Good agreement between model simulations and experimental data was achieved.     

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.     

Tolerance and stress response of sclerotiogenic <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> G15 to copper and lead

Aspergillus oryzae G15 was cultured on Czapek yeast extract agar medium containing different concentrations of copper and lead to investigate the mechanisms sustaining metal tolerance. The effects of heavy metals on biomass, metal accumulation, metallothionein (MT), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were evaluated. Cu and Pb treatment remarkably delayed sclerotial maturation and inhibited mycelial growth, indicating the toxic effects of the metals. Cu decreased sclerotial biomass, whereas Pb led to an increase in sclerotial biomass. G15 bioadsorbed most Cu and Pb ions on the cell surface, revealing the involvement of the extracellular mechanism. Cu treatment significantly elevated MT level in mycelia, and Pb treatment at concentrations of 50–100 mg/L also caused an increase in MT content in mycelia. Both metals significantly increased MDA level in sclerotia. The variations in MT and MDA levels revealed the appearance of heavy metal-induced oxidative stress. The activities of SOD, CAT, and POD varied with heavy metal concentrations, which demonstrated that tolerance of G15 to Cu and Pb was associated with an efficient antioxidant defense system. In sum, the santioxidative detoxification system allowed the strain to survive in high concentrations of Cu and Pb. G15 depended mostly on sclerotial differentiation to defend against Pb stress.