2,5-DKG还原酶Ⅰ的分离纯化与性质研究

欧文氏菌ER97高效表达了从棒状杆菌SCB3058克隆的2,5-二酮基-D-葡萄糖酸（2,5-DKG）还原酶Ⅰ基因,5L罐发酵后,收集菌体破碎,将胞内可溶性的蛋白通过硫酸铵分级沉淀、DEAE—Sepharose CL-6B离子交换柱层析和Phenyl Sepharose CL-4B疏水柱层析后分离纯化到了2,5-DKG还原酶Ⅰ,纯化了5倍,得率27％,比活力为3,418U／mg。测定了该酶的一些特性参数：分子量为34kD,等电点为6．0,它以NADPH为辅酶,将2,5-DKG还原为2-酮基-L-古龙酸（2-KLG）,对NADPH和2,5-DKG底物的Km值分别是0．29mmol／L和14．7mmol／L,1mmol／L Cu^2＋、Zn^2＋等有强烈抑制作用,EDTA和巯基乙醇对该酶没有抑制作用,酶的最适pH为7．0,最适反应温度为40℃。     

链霉菌Str s-2产木聚糖酶的条件及部分性质研究

通过碳氮源对链霉菌Str s-2产胞外木聚糖酶活性的影响,得出其适宜培养基为(g/L)：含半纤维素20,(NH4)2SO4 4．0,KH2PO4 1．0,MgSO4-7H2O 0．5,NaCl 0．3,CaCO3 1．0。用DNS法研究了该酶的性质结果表明其最适pH值为6．5,最适反应温度为55℃;Na^ 、K^ 、Ca^2 、Mg^2 等离子对酶有激活作用,而Zn^2 、Ag^ 、Fe^3 和Cu^2 离子则抑制酶的活性。     

硫营养对重金属胁迫下玉米和小麦根系导水率的影响

以‘秦单4号’玉米和‘小偃22’小麦为供试材料,通过室内玉米水堵和小麦盆栽试验,采用静态压力室法测定了不同硫营养水平与不同重金属胁迫处理下根系导水率（Lpr）的变化。结果表明：玉米Lpr经100μmol／LHg^2＋、Zn^2＋、Cu^2＋胁迫处理后分别降低到CK的13％、79％和45％,Cu^2＋、Zn^2＋对其Lpr的抑制效应分别为同浓度Hg^2＋的62．9％及24．3％;施硫处理的玉米Lpr在无Cu^2＋胁迫条件下都极显著高于无硫处理,且低硫处理显著较高,在Cu^2＋胁迫条件下亦都极显著高于无硫处理,并且Cu^2＋胁迫对其Lpr的抑制效应随着硫浓度增大而逐渐减小;不施硫和施硫处理的小麦Lpr在500mg／kgZn^2＋胁迫下分别比相应CK降低36．89％和37．71％,在400mg／kgCu^2＋胁迫下则分别比相应CK降低了34．61％和12．29％。研究发现,重金属Cu^2＋、Zn^2＋对玉米和小麦根系导水率具有显著的抑制作用且Cu^2＋〉Zn^2＋,施硫对Cu^2＋胁迫下的玉米和小麦根系导水率都具有显著的保护作用,并随硫浓度增加而增强。     

水杨酸提高香蕉幼苗的抗冷性与H2O2代谢有关

探讨了水杨酸(salicylic acid,SA)提高香蕉幼苗抗冷性的可能机理。在常温下(30／22℃)用不同浓度(0—3．5mmol／L)的SA水溶液喷洒叶片1d,置于7℃低温下冷胁迫3d,随后于常温下恢复2d后测定电解质泄漏率,结果表明：SA0．3～0．9mmol／L能显著提高香蕉幼苗的抗冷性,以0．5mmol／L效果最佳。若把冷胁迫温度降到5℃,SA0．5mmol／L预处理可显著减少幼苗叶片的萎蔫面积。但当SA浓度高于1．5mmol／L时,恢复期间的电解质泄漏甚至高于对照(蒸馏水处理),表明它们加剧了冷害。SA提高香蕉幼苗的抗冷性可能需要H2O2的参与：1)SA0．5mmol／L常温处理诱导了H2O2的积累和活性氧造成的膜脂过氧化——三氯乙酸反应物质(TBARS)的增加,这可能与H2O2的清除酶——过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性的受抑和H2O2的产生酶—超氧化物歧化酶(SOD)活性几乎不受影响有关;2)外源H2O2(1．5—2．5mmoL／L)也能显著降低低温胁迫期间的电解质泄漏,表明也能提高抗冷性;3)而用H2O2的捕捉剂——二甲基硫脲(DMTU)可明显抑制SA诱导的抗冷性;4)在低温胁迫与恢复期间,SA预处理明显提高了CAT和APX的活性,抑制了H2O2与TBARS的快速上升。     

大肠杆菌发酵生产SOD最佳条件研究

通过对大肠杆菌进行液体发酵培养,用正交实验探索了四种微量元素（Cu^2 ,Zn^2 ,Mn^2 ,Fe^2 )加入量,培养时间,摇床转速和培养温度等对大肠杆菌细胞增养生产SOD的影响,最佳工艺条件为：CuSO450μmol/L,ZnSO430μmol/L,MnSO450μmol/L,FeSO430μmol/L,培养时间14h,摇床转速200r/min,培养温度38℃,产酶量7726IU/g湿菌体。     

牛血清白蛋白对超氧化物歧化酶的化学修饰

目的：通过化学修饰提高超氧化歧化酶（SOD）的稳定性,考察金属离子在不同浓度下对SOD活性的影响。方法：用戊二醛作为交联剂,用牛血清白蛋白（BSA）将牛红细胞超氧化物歧化酶进行化学修饰,得到SOD的修饰酶。对比研究三种SOD：修饰酶,混合酶及天然酶的理化性质。结果：修饰酶等电点降低,对温度、pH的稳定性较天然酶有很大提高,对胰蛋白酶和胃蛋白酶也表现出很强的耐水解性。二价离子Mg^2 、Mn^2 对天种SOD活力均有不同程度的抵制作用,Ca^2 、Zn^2 、Cu^2 对修饰酶活力有激活作用,一价离子K^ 对三种OSD活力均无明显影响.结论:修饰酶较天然酶的稳定性有很大的提高,加入Ca^2 、Zn^2 、Cu^2 可提高修饰酶的活力。     

荔枝果皮过氧化物酶的纯化及部分酶学性质研究

经硫酸铵分级盐析、DEAE-Sepharose和Sephadex G-75柱层析分离,从荔枝果皮中分离提纯了过氧化物酶(POD),该酶被纯化了12．5倍,产率为1．9％。经SDS-PAGE确定为单一条带。该酶最适反应温度为35℃,对热具有较强的稳定性,经75℃处理30min,酶活性只损失50％。最适pH约为6．5,但在pH4．0—8．0范围内活力仍比较稳定。该酶在25℃和0．05mol／L磷酸缓冲液(pH7．0)条件下对愈创木酚、邻苯二酚和没食子酸的Km分别是2．75、12．4和12．8mmol／L。二硫苏糖醇和抗坏血酸能完全抑制POD活性,L-半胱氨酸、柠檬酸、FeS04、GSH、SDS和ZnS04对POD活性有一定的抑制作用,而FeCl,和CuSOt对POD则有较好的激活作用。     

均匀设计优化建兰ISSR-PCR体系

采用均匀设计,对影响建兰ISSR-PCR体系的引物、Mg^2＋、dNTP和Taq DNA聚合酶浓度等进行4因素5水平和4因素3水平两轮优化,建立了适合于建兰ISSR-PCR的反应体系：在20μL反应体系中,舍引物0．25μmol／L、Mg^2＋ 2．5mmol／L,dNTP0．2mmol／L、Taq DNA聚合酶1.5U和模板DNA40ng．在此基础上对扩增程序中的循环次数和退火温度,以及ISSR引物进行筛选．筛选获得的扩增程序为：94℃预变性5min;接着进行32个循环：94℃变性35S,52～56℃退火45S,72℃延伸90s;循环结束后,72℃延伸10min．同时筛选得到14个扩增稳定、多态性丰富的ISSR引物．     

荧光素酶基因luc在大肠杆菌中表达条件优化

对重组荧光素酶大肠杆菌菌株M15／pQE30-luc进行了表达条件的优化研究。单因素结果表明：在初始pH值7．0,装液量为20％,2％的接种量,终浓度为0．5mmol／L的IPTG,添加10—30mmol／L的Mg^2＋,摇床转速为200r／min,37℃诱导3．5h酶的表达量最高。正交试验结果表明：初始pH值为7．0,添加40mmol／LMg^2=,接种量2％,装液量为20％时表达量最高,比酶活达1．63×10^8RFU／mg蛋白。     

青霉素敏感的酶场效应晶体管的研究和应用

酶场效应晶体管(ENFET)是近年来发展起来的一类半导体技术和生物技术相结合的新型器件。双管差分输出式ENFET对环境温度变化及总体pH变化等外界因素有自动补偿作用,其输出电压随时间的漂移较之单管输出有很大的改善。此外,还可采用金电极作参比电极以实现探头的小型化。青霉索ENFET在0．01mol／L和O．02m01／L的磷酸缓冲液中的响应灵敏度分别为6．5—7．0mV／mmol／L和3．2—3．6mV／mmol／L,线性范围为0．5—14mmol／L和0．5—25mm0l／L。当浸于O．Olmol／L磷酸缓冲液、置于4℃下保存时,探头的贮存寿命大于6个月。探头的累计使用次数可超过1000次。本文还论述了青霉素ENFET在青霉素发酵液浓度 测定中的应用。     

Relationship between melanin content and superoxide dismutase (SOD) activity in the liver of various species of animals

The scavenger effect of melanin and of superoxide dismutase (SOD) activity on superoxide anion has been shown. In this work we show the relationship between melanin content and SOD activity in livers containing different quantities of melanin which were taken from various species of animals. The mitochondrial SOD activity disappears when the melanin content in the liver is very high; moreover it increases, in the liver of various species of animals examined, proportionally to the decrease of melanin content. No significant variation of the SOD activity localized in the soluble fraction has been detected when related to the melanin content. We think that in the pigmented liver the antioxidant activity of the melanin could mimic part of the function of SOD. The loss of Mn SOD activity could be mediated by a low intracellular level of superoxide anion due to the scavenger effect of melanin on superoxide anion; in fact, it is well known that the biosynthesis of Mn SOD is induced by intracellular levels of superoxide anion.     

Exposure to anoxia of the clam, Chamelea gallina: II: Modulation of superoxide dismutase activity and expression in haemocytes

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity, clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper, activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of C. gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure), SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values.In the first experiment (24 h anoxia), reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls. The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore, the high values of activity shown by Mn-SOD during the recovery are likely to be due to the high inducibility of this isoform.In Cu/Zn-SOD expression analyses, two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa) corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.     

沙蜇(Stomolophus meleagris)刺丝囊毒素生物活性的初步分析

从沙蜇触手提取刺丝囊细胞毒素,并对该毒素进行溶血活性、致死活性、SOD活性和抗肿瘤活性的研究。结果显示,沙蜇毒素具有明显的溶血活性,其半溶血率(HU50)约为10.5μg/ml;该毒素还对草鱼显示出较强的致死活性,半致死量(LD50)为50μg毒素/g鱼;同时该毒素具有明显的SOD活性和抗肿瘤活性,当毒素浓度为18μg/ml时其总SOD活性为161 U/mg,而毒素浓度为1 mg/ml时,该毒素对肝癌细胞Bel-7402表现出显著的抑制效果,其抑制率达到54.9%。因此,有必要对沙蜇毒素内的生物活性组分进行深入研究,为沙蜇毒素的开发利用提供依据。     

Changes in serum markers indicative of health effects in vineyard workers following exposure to the fungicide mancozeb: an Italian study

Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical–pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.     

SOD制剂在椪柑生产上的应用试验

本试验初步探明喷施益微SOD制剂,对椪柑成年结果树的果实SOD活性及品质有良好效应,建议采用0.16g/L SOD制剂进行喷施,每隔30d一次,连续喷布3~4次较为合适。     

Superoxide dismutase as a target enzyme for Fe-porphyrin-induced cell death

The cell viability of human cancer cell lines treated with [5,10-bis(N-methyl-4-pyridyl)-15,20-diphenyl]porphinatoiron(III) (cis-FeMPy(2)P(2)P) has been estimated. The cis-FeMPy(2)P(2)P is a superoxide dismutase (SOD) mimic in vitro that exhibited a significant toxicity in cancer cell lines. This toxicity is rather due to pro-oxidant properties of the iron-porphyrin in vivo. We have demonstrated that there was the relationship between the LD(50) values calculated from the viability of cancer cell lines treated with cis-FeMPy(2)P(2)P and the SOD activities of the cell lines. Furthermore, the inhibition of SOD by antisense S-oligonucleotide increased the cytotoxic effect of cis-FeMPy(2)P(2)P against cancer cells. These results suggest that SOD is a target enzyme for the cell death induced by cis-FeMPy(2)P(2)P as a new class of anticancer agents.     

Phytochemical screening of flavonoids with their antioxidant activities from rapeseed (Brassica napus L.)

Ten flavone compounds, including three new flavonoid glycosides, were isolated from defatted rapeseed, and their protective antioxidant effect on H₂O₂-induced oxidative damage in human umbilical vein endothelial cells (ECV-304) was investigated. Three new flavonoid glycosides were identified as kaempferol-3-O-[(6-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (8), kaempferol-3,7-di-O-β-d-glucopyranoside-4'-O-(6-O-sinapoyl)-β-d-glucopyranoside (9), and kaempferol-3-O-[(3-O-sinapoyl)-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranoside]-7-O-β-d-glucopyranoside (10). The protective effects of all of the isolated compounds on H₂O₂-induced oxidative damage were assessed, and the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) were measured. All of compounds had a protective effect on H₂O₂-induced oxidative damage in ECV-304 cells and the presence of a substituted sinapoyl group and its position in the structures were used to elucidate the activity differences.     

海藻糖对微生物谷氨酰氨转胺酶热稳定性研究

本实验目的是研究海藻糖对微生物谷氨酰胺转胺酶（TGase）热稳定性的作用。糖类对TGase的保护作用根据糖种类不同有所差异,海藻糖和蔗糖的保护作用优于葡萄糖对TGase的保护作用。在45℃、50℃、55℃、60℃、65℃下研究了海藻糖对TG酶的保护作用。结果表明,在50～65℃下海藻糖使谷氨酰胺转胺酶受热时的稳定性提高了约20％。海藻糖与酶复合的最合适浓度约为14％,浓度低时保护作用不明显,加入过高浓度的糖对酶的活性维持不利。50℃下处理一段时间内,海藻糖对酶的保护作用随时问变化很小。     

超氧物歧化酶（SOD）与柳树分布相关性的研究

在研究红松苗越冬伤害原因时,发现超氧物歧化酶(SOD),在防止针叶越冬伤害中起着重要作用。75%术条遮荫,可安全越冬的红松苗针叶,早春仍保持较强的活性,脂质过氧化产物积累较少。而暴露于全光下,则终于发生不可逆伤害,红松苗针叶SOD活性低而脂质过氧化产物(丙二醛)积累较多。并且暴露在全光下不出现伤害的红松大树针叶,酶活性也高于幼苗。此外,关于SOD对植物在低温、干旱、日灼、臭氧、大气污染(SO_2)等逆境中的抗性作用,也已有不少报道。日本学者曾     

发菜中超氧化物歧化酶基因的克隆及在大肠杆菌中的表达

采用基因工程技术从发菜总DNA中克隆了一段的基因序列,该序列与基因库中已公布的编码地木耳（Nostoc commnue）超氧化物歧化酶的氨基酸序列同源性为97%。将该基因插入含T7启动子质粒pET-32中构建表达质粒pET-sod,然后将该表达质粒转入大肠杆菌BL21中进行蛋白表达,表达菌株用1 mmol·L^-1 IPTG诱导表达数小时后,产生较多的重组的蛋白,且该蛋白以可溶性蛋白形式存在。SDS-PAGE分析表明,在相对分子量约为22 kd的位置有一条明显蛋白质带。将诱导表达后的蛋白通过亲和层析的方法进行蛋白纯化;NBT光还原法测定表达产物的比活力,每毫克纯化蛋白约为2 550 U。对纯化后的蛋白进行高温胁迫研究,将该纯化蛋白在60℃高温下胁迫90 min后,其活性为原（未经胁迫）蛋白活性的85%。