压力作用下面包酵母胞内谷胱甘肽和麦角固醇的变化

以高纯空气（O2∶N2 ＝21∶79）为加压介质,研究了0.5Mpa压力下面包酵母CICC1447和CICC1339细胞生长及细胞内谷胱甘肽、麦角固醇的含量变化。结果表明：加压培养时两株酵母菌的对数生长期延迟出现,对数生长期的持续时间缩短,而且两株菌的比生长速率均明显低于对照组,同时两株菌的倍增时间也较对照组有所延长;压力刺激可显著提高面包酵母细胞内谷胱甘肽（GSH）的含量,但麦角固醇含量的变化却不明显。在0.5MPa压力下保压培养3h时CICC1447胞内谷胱甘肽含量比常压对照组提高了42.6%,而加压3h后麦角固醇含量比对照组提高了20.1％;加压培养6h时CICC1339胞内谷胱甘肽含量较对照组提高了58.7%,但其麦角固醇含量反而降低。这说明不同的酵母菌对压力刺激的反应是不同的。     

蛹虫草液体发酵产多糖的条件优化

多糖是蛹虫草Cordyceps militaris产生的重要活性成分。为提高其液体发酵胞外多糖的产量,通过单因素轮换法、方差分析、正交试验对蛹虫草菌株YCC-H产胞外多糖的发酵条件进行了优化。单因素结果表明液体发酵最适初始pH为5,最适葡萄糖浓度(质量分数)为6%,最适氮源为酵母膏,最适装液量为80 mL/250 mL,最适接种量(体积分数)为12%;在不同培养条件下菌体生物量变化较大;通过方差分析及正交试验进一步优化,结果显示：装液量(A)、氮源(B)、接种量(C)对蛹虫草产胞外多糖影响程度为B>A>C,最佳条件组合为A₂B₃C₂,即装液量80 mL/250 mL、酵母膏为唯一氮源、接种量(体积分数)为12%,其余因素选择单因素中最佳水平。经验证,该菌株合成胞外多糖的质量浓度达247.06 mg/L,较未优化前胞外多糖的产量23.67 mg/L提高了9.43倍。     

谷胱甘肽与铜绿假单胞菌致病性的关系

铜绿假单胞菌由于对抗生素的固有耐药和多重抗药性, 已成为医院内感染的重要病原菌之一。谷胱甘肽是细胞内最重要的抗氧化剂, 保护细胞免受氧化压力的损害。但是在铜绿假单胞菌感染的组织中, 由于绿脓菌素等致病因子的存在, 可以导致谷胱甘肽的水平降低。同时, 谷胱甘肽又可以增强绿脓菌素的致病性。本文就谷胱甘肽与铜绿假单胞菌关系的最新研究进展并结合作者的工作, 对上述问题进行了综述和探讨。     

大肠杆菌NAD+合成关键酶的克隆表达及发酵优化

【目的】烟酰胺腺嘌呤二核苷酸(NAD~+)在细胞基因表达、氧化还原反应、能量代谢以及调控细胞生命周期中具有重要的作用,其细胞内含量是能量效率的关键因素。强化辅因子合成策略,获得高产NAD~+菌株,对于NAD~+依赖型氧化还原反应的速率和调节相关生化合成途径的代谢流具有重要意义。【方法】首先通过内源性调节,对代谢途径中的关键酶基因进行强化,过量表达和共表达NAD~+合成途径中的关键酶基因pncB、nadD和nadE;其次,通过外源调节增加NAD~+前体物,优化诱导条件提高发酵过程中关键酶的表达量,增加NAD~+的合成量;最后在单因素优化试验的基础上,以NAD~+含量为响应值,采用Box-Bohnken试验设计方法,研究3个显著性影响因素相互作用对NAD~+积累量的影响,确定最佳的优化条件。【结果】根据关键酶基因强化策略,构建了7株重组菌,其中重组菌E.coli BL21/p ET-21a-nad E-pncB胞内NAD~+含量相比初始菌株E.coli BL21/pET-21a提高了405.2%。通过对该菌株诱导条件和NAD~+合成前体的优化,使用Design Expert 8.0分析实验数据,得出该重组菌株的最佳发酵条件为:诱导温度控制在15–20 oC,OD_(600)为0.6–0.8时添加IPTG 0.63 mmol/L、烟酸15.8 mg/L、诱导时长控制在24 h。NAD~+含量在最优条件下实验验证值可达43.16μmol/g DCW,与优化前相比提高了123.6%,与初始菌株相比提高了1029.8%。【结论】在大肠杆菌中共表达关键酶基因pncB和nadE,胞内NAD~+合成量明显增加,前体物以及诱导条件的外源调节使NAD~+积累量达到最佳优化值。实现了提高NAD~+含量的目标,胞内辅因子浓度的增加为提高生物催化效率奠定了可行性基础。     

海洋来源担子菌产葡萄糖氧化酶发酵条件优化

为提高从大连渤海海域海泥中筛选到的菌株Basidioascus sp. LG-31产低温葡萄糖氧化酶活性性,对菌株Basidioascus sp.LG-31进行了单因素发酵条件优化,结果表明,菌株Basidioascus sp.LG-31产低温葡萄糖氧化酶活性显著提高。单因素优化结果：蔗糖4%(质量分数)、酵母粉0.3%(质量分数)、NaNO₃ 0.4%(质量分数)、无机盐(KH₂PO₄ 0.02%(质量分数)、KCl 0.10%(质量分数)、MgSO₄·7H₂O 0.10%(质量分数))、初始pH值7.0、装液量125 mL/250 mL、接种量2%(体积分数)、转速200 r/min、温度20℃。最高酶活性达435.37 U/mL,相对于优化前的最高酶活性15.28 U/mL,提高了28.49倍。为下一步酶学性质研究及低温葡萄糖氧化酶在工业中的应用提供参考。     

ε-多聚赖氨酸和谷胱甘肽影响抗真菌药物对烟曲霉作用效果

为了解多肽ε-多聚赖氨酸和谷胱甘肽对常见抗真菌药物体外抗人类病原真菌烟曲霉效果的影响,利用平板点菌实验、最低抑菌浓度实验中的微量液基法和E-test法,以及ROS检测等方法研究ε-多聚赖氨酸和谷胱甘肽在抗真菌药物体外抗烟曲霉过程中的作用以及可能的作用机制。通过基因定点敲除谷胱甘肽合成酶基因gcsA和gshA观察细胞内谷胱甘肽对于烟曲霉生长的重要性。结果显示,ε-多聚赖氨酸与抗真菌药物尤其是卡泊芬净和伊曲康唑呈协同作用;而谷胱甘肽则能够明显地拮抗伊曲康唑和卡泊芬净的抗烟曲霉作用。ε-多聚赖氨酸与伊曲康唑联用时菌体内的ROS含量比单独使用伊曲康唑时显著增多,而谷胱甘肽与伊曲康唑联用时菌体内的ROS含量比伊曲康唑单独使用时减少。基因敲除实验结果显示γ-L-谷氨酰-L-半胱氨酸合成酶基因gcsA为烟曲霉生长必需基因,而谷胱甘肽合成酶基因gshA对生长没有明显影响。结果表明,ε-多聚赖氨酸与谷胱甘肽影响了伊曲康唑和卡泊芬净对烟曲霉的作用。ε-多聚赖氨酸通过促进伊曲康唑刺激细胞产生ROS诱导烟曲霉死亡从而实现协同作用。谷胱甘肽为细胞内必需还原型多肽,通过消除胞内伊曲康唑诱导产生的ROS从而产生拮抗作用。     

废弃活性污泥中产聚羟基脂肪酸酯菌株Bacillus sp. PB-3的发酵条件优化

通过尼罗红染色法结合荧光显微镜镜检,从废弃活性污泥中分离得到1株高产聚羟基脂肪酸酯(PHAs)的菌株Bacillus sp.PB-3,经气相色谱法鉴定该菌株胞内产物为聚β-羟基丁酸酯(PHB)。对培养基成分及发酵条件优化后,获得最佳培养方案:12 g/L的葡萄糖为C源,2 g/L的牛肉膏为N源,初始pH 7.5,培养基装液量80 mL,转速为200 r/min,37℃培养48 h,PHB质量分数可达菌体干质量的32.09%,比优化前提高30%。     

CydDC蛋白的表达及对谷胱甘肽运输的影响

在重组大肠杆菌中共表达了谷胱甘肽运输蛋白CydDC与双功能合成酶GshF,通过增强对谷胱甘肽的运输作用,提高谷胱甘肽产量。实验结果表明,目的蛋白CydDC及GshF均成功表达;重组菌MG1655(pTrc99a-as/p BAD33-cydDC)总谷胱甘肽产量为0.22 mmol/L,胞外谷胱甘肽含量显著增加,达到81.9%,分别是对照菌MG1655(pTrc99a-as/pBAD33)的1.11倍和1.29倍。     

谷胱甘肽对铜绿假单胞菌exoS 和exoY基因的影响

摘要: 【目的】研究谷胱甘肽对铜绿假单胞菌exoS和exoY基因表达的影响。【方法】利用丁硫氨酸亚砜胺和马来酸二乙酯同时耗竭细胞内的谷胱甘肽,并构建包含被lacZGm破坏的谷胱甘肽合成酶基因的突变体。通过分别连有exoS 和exoY基因启动子的pMS402质粒上Lux报道子发光值大小检测exoS 和exoY基因表达变化情况。【结果】exoS和exoY基因的表达在用化学药品耗竭的细胞中或是在谷胱甘肽合成酶突变体中都降低。【结论】铜绿假单胞菌细胞内的谷胱甘肽可以促进exoS和exoY的表达。这将为进一步研究铜绿假单胞菌的感染以及致病性机理提供一定的理论基础。     

酵母生产谷胱甘肽的培养条件研究

应用Plackett-Burman实验设计、响应面分析方法研究了酵母生产谷胱甘肽的培养条件,结果表明:最佳培养条件为初始pH 5.0,培养温度28℃,接种量10%,摇床转速200 r/min,种子液种龄22~23 h。葡萄糖1.95%,糖蜜1.95%,蛋白胨3%,Cys.HCl 0.10%,MgSO4.7H2O 0.5%,甲硫氨酸0.05%,在此优化的条件下培养,谷胱甘肽的产量达235.7 mg/L,比优化前提高45.4%。     

A new strategy to enhance glutathione production by multiple H2O2 induced oxidative stresses in Candida utilis

Effect of H(2)O(2)-induced oxidative stress on glutathione (GSH) production in Candida utilis was investigated. Based on the results that H(2)O(2) can effectively stimulate GSH accumulation but inhibit cell growth simultaneously, a novel strategy of multiple H(2)O(2) stresses with different concentrations (1 mmol/L at 4h, 2 mmol/L at 8h, and 4 mmol/L at 12h) were developed to maximize GSH production. As a result, a maximal GSH yield of 218 mg/L was achieved and a corresponding intracellular GSH content was 2.15%, which were 54.6% and 58.1% higher than the control. By further applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 328 mg/L and 2.30%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H(2)O(2).     

Antioxidative response of ascorbate-glutathione pathway enzymes and metabolites to desiccation of recalcitrant Acer saccharinum seeds

Ascorbate–glutathione systems were studied during desiccation of recalcitrant seeds of the silver maple (Acer saccharinum L.). The desiccated seeds gradually lost their germination capacity and this was strongly correlated with an increase in electrolyte leakage from seeds. Simultaneously the increase of reactive oxygen species (ROS) (superoxide radical – O₂⁻ and hydrogen peroxide – H₂O₂) production was observed. The results indicate that remarkable changes in the concentrations and redox status of ascorbate and glutathione occur in embryo axes and cotyledons. After shedding, concentrations of ascorbic acid (ASA) and the reduced form of glutathione (GSH) are higher in embryo axes than in cotyledons and their redox status is high in both embryo parts. Cotyledons in freshly shed seeds are devoid of GSH. At the first stages of desiccation, up to a level of 43% of moisture content, ASA content in embryo axes and GSH content in cotyledons increased. Below this level of moisture content, the antioxidant contents as well as their redox status rapidly decreased. The enzymes of the ascorbate–glutathione pathway: ascorbate peroxidase (APX) (EC 1.11.1.11), monodehydroascorbate reductase (MR) (EC 1.6.5.4), dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) increased their activity during desiccation, but mainly in embryonic axes. The changes are probably required for counteracting the production of ROS during desiccation. The relationship between ascorbate and glutathione metabolism and their relevance during desiccation of recalcitrant Acer saccharinum seeds is discussed.     

Protective effect of glucose-cysteine adduct on thein situ perfused rat liver

Summary Insitu perfusion of rat liver was performed with a medium containing glucose-cysteine adduct [2-(D-gluco-pentahydroxypentyl) thiazolidine-4-carboxylic acid, glc-cys] and its effect on glutathione (GSH) and ATP levels and bile production was examined. The GSH content in the liver was maintained at the original level during perfusion with 1 mM glc-cys for 2h, while it decreased significantly in the absence of glc-cys. After 4h of perfusion without glc-cys, ATP content and bile production decreased significantly besides the decrease in GSH content, but they were maintained at the original levels with glc-cys. When the perfusion was performed with the liver of rats injected with diethyl maleate (DEM), the GSH level, which was decreased to 6.0% of the control by DEM injection, was restored to 22.6% of the original level by perfusion with 2mM glc-cys for 30 min. Data indicate that glccys is a cysteine prodrug with protective action on the liver.     

Liver cancer is induced by a subnecrogenic dose of DENA when associated with fasting/refeeding: role of glutathione-transferase and lipid peroxidation.

In previous studies, we reported that fasting/refeeding has a role in sustaining the initiation of liver cancer by a subnecrogenic (noninitiating) dose of diethylnitrosamine (DENA). This research investigated whether the metabolic alterations imposed by fasting/refeeding provide an imbalance between the generation of carcinogenic molecules and the scavenger defense mechanisms in rat liver. Metabolism of DENA, levels of reduced glutathione (GSH) and GSH transferase (GST) activity, as well as basal and stimulated malondialdehyde (MDA) production, were examined. Rats fasted for 4 days showed a decrease in the liver levels of GSH, GST activity, monounsaturated fatty acids and % of labeled nuclei. After 1 day of refeeding, at which point DENA was administered, the levels of GSH recovered, GST activity remained below control values, basal and stimulated MDA production and content of total polyunsaturated fatty acids in liver phospholipids decreased. One day after DENA treatment, MDA production further decreased, although the % of labeled nuclei increased. No significant changes in the content of arachidonic acid, the main target of peroxidation, were observed at any time. The results indicated that the induction of the hepatocellular carcinoma was associated with a depression of GST activity and lipid peroxidation when rats were given 20 mg/kg of DENA after 1 day of refeeding after 4-day fasting.     

Salt-induced osmotic stress for glutathione overproduction in Candida utilis

The effect of NaCl-induced osmotic stress on glutathione (GSH) production was investigated in Candida utilis. Based on the fact that NaCl stress can enhance GSH production but inhibit cells growth simultaneously, the novel strategies of multiple osmotic stresses with different NaCl additions (0.2 mol/l at 4 h, 0.4 mol/l at 8 h, and 0.6 mol/l at 12 and 16 h) were developed for GSH overproduction. After 30 h cultivation, GSH yield reached 238 mg/l and intracellular GSH content was 2.34%, increased by 66.4% and 70.7% respectively compared to the control. Further applying the strategies to 7 l fermentor, GSH yield of 356 mg/l was achieved at 30 h, which was 65.6% higher than the control. Moreover, NaCl stress led to an increase in intracellular cysteine content and activities of γ-glutamylcysteine synthetase, GSH synthetase and GSH reductase, explaining the mechanism involved in inducing cellular GSH accumulation.     

Intracellular adaptations of glutathione content in Cucurbita pepo L. induced by treatment with reduced glutathione and buthionine sulfoximine

The intracellular effects of GSH (reduced glutathione) and BSO (buthionine sulfoximine) treatment on glutathione content were investigated with immunogold labeling in individual cellular compartments of Cucurbita pepo L. seedlings. Generally, GSH treatment led to increased levels of glutathione in roots and leaves (up to 3.5-fold in nuclei), whereas BSO treatment significantly decreased glutathione content in all organs. Transmission electron microscopy revealed that glutathione levels in mitochondria, which showed the highest glutathione labeling density of all compartments, remained generally unaffected by both treatments. Since glutathione within mitochondria is involved in the regulation of cell death, these results indicate that high and stable levels of glutathione in mitochondria play an important role in cell survival strategies. BSO treatment significantly decreased glutathione levels (1) in roots by about 78% in plastids and 60.8% in the cytosol and (2) in cotyledons by about 55% in the cytosol and 38.6% in plastids. After a short recovery period, glutathione levels were significantly increased in plastids and the cytosol of root tip cells (up to 3.7-fold) and back to control values in cotyledons. These results indicate that plastids, either alone or together with the cytosol, are the main center of glutathione synthesis in leaves as well as in roots. After GSH treatment for 24 h, severe ultrastructural damage related to increased levels of glutathione was found in roots, in all organelles except mitochondria. Possible negative effects of GSH treatment leading to the observed ultrastructural damage are discussed.     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.     

Differences in glutathione oxidation and transpeptidylation between normal liver and hepatomas (author's transl)

Total homogenates from liver tissues, as well from Morris 3924 A and Yoshida AH-I30 hepatomas display a different degree of thiobarbituric acid reacting substances (TBArs) when incubated "in vitro". It is well known that carbonyl compounds arising from lipoperoxidative decomposition of unsaturated fatty acids can easily react with reduced glutathione (GSH). So, the decay in GSH we have shown in previous experiments could be accounted for GSH trapping by the formed aldehydes. Some discrepancies were, however, seen when the decay in GSH and the increase in GSSG were compared, both in normal and in tumour tissues. It is known that GSH can be destroyed not only through oxidative process, but also through the action of gamma-glutamyl-transpeptidase. In the present paper the decrease of total (TG) and reduced (GSH) glutathione was followed and compared with both the increase in GSSG and the increase in the production of TBArs, during "in vitro" incubation. In normal liver, increase in TBArs production parallels the decay in GSH concentration; GSSG, on the contrary, increases. In AH-I30 Yoshida hepatoma cells, TBArs production is lower and GSSG is also decreased. In 3924 A Morris hepatoma GSH decrease is similar to that observed in the liver, while TBArs production is lower and GSSG is also decreased. Analysis of TG content during the incubation-time suggests that GSH decay in both hepatoma types is essentially due to gamma-glutamyl-transpeptidase action, whilst GSH oxidation to GSSG is decreased.