聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

β—葡聚糖酶的分离纯化和特性研究

对里木霉所产β－葡聚糖酶粗酶液通过饱和硫酸铵沉淀、Sephadex G-100柱层析和DEAE－Sephadex A-50柱层析进行纯化,比活提高14．60倍,活力回收6．62％。酶特性研究表明,最适温度和pH分别为60℃和5．0,在pH低于5．0时酶较稳定,酶的热稳定性在60℃以下。Cu^2 、Mn^2 、Mg^2 、Fe^3 和K^ 对酶有抑制作用,Zn^2 、Ca^2 、Co^2 和Fe^2 有激活作用。     

重组定点突变巴曲酶酶学性质研究

目的对重组定点突变巴曲酶的酶学性质进行研究,为开发成临床用药奠定基础。方法测定不同的温度、pH缓冲液和金属离子等条件对重组定点突变巴曲酶活性的影响。结果重组定点突变巴曲酶的最适pH值在6．5～7．5之间。该酶在50℃以下活力保持90％以上,但当温度超过60℃时,该酶已完全失活。Ca^2＋和Na^＋离子对酶的稳定性无明显影响,而Mg^2＋、K^＋、Mn^2＋离子则表现为激活作用,Zn^2、＋Cu^2＋、Fe^2＋、Co^＋离子则表现为明显的抑制作用。结论重组定点突变巴曲酶在中性条件下比较稳定,它不耐高温,金属离子对其活性有一定的影响。     

重金属诱导拟南芥原生质体DNA损伤的单细胞凝胶电泳检测

以拟南芥原生质体为实验体系,研究不同浓度的3种重金属离子对拟南芥原生质体的毒性和DNA损伤的差异。结果表明,用1-5mmol·L^-1的Zn^2＋、Cd^2＋和Cu^2＋分别处理的拟南芥原生质体,2小时内活力逐渐下降,并表现出明显的浓度依赖性：与相同浓度的Cd^2＋和Cu^2＋相比,Zn^2＋对拟南芥原生质体活力的影响程度较小,表现出较低的毒性。单细胞凝胶电泳检测发现,用0．1-0．8mmol·L^-1的Zn^2＋、Cd^2＋和Cu^2＋分别处理拟南芥原生质体30分钟,以OTM值表示的原生质体DNA损伤量随重金属离子浓度的增加而递增：相同浓度（0．5mmol·L^-1）的3种重金属离子相比,Zn^2＋对原生质体的遗传毒性明显低于Cu^2＋和Cd^2＋。综合原生质体活力和DNA损伤的单细胞凝胶电泳检测结果,发现ZnO^2＋对拟南芥原生质体的遗传毒性较低,而CdO^2＋和Cu^2＋的遗传毒性较高。本研究建立的拟南芥原生质体实验体系,结合运用单细胞凝胶电泳技术,能够快速、灵敏地检测重金属对植物细胞的遗传毒性。     

牛血Cu/Zn-SOD的热稳定性

研究了不同的Cu^2 和Zn^2 浓度配比对SOD热稳定性的影响,并测定了SOD活性及利用考马斯亮蓝D250法测定蛋白质含量。结果表明在Cu^2 、Zn^2 分别为5．4mmol／1．和3．6mmol／L时SOD的活性最高,达到了92．6％;在75℃时大部分杂蛋白变性沉淀,SOD的比活力最高。最后确定在加入5．4mmol／L．Cu^2 ,3．6mmol／LZn^2 条件下,75℃下热变性,可以取得比较高活性和比活性的SOD。从而为寻找更简便、经济的提纯方法提供参考依据。     

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。     

修饰血红素提高血红蛋白类过氧化物酶活性

利用苯酚或对羟基联苯对血红蛋白的血红素辅基进行化学修饰,将修饰后的血红素与脱辅基血红蛋白进行重组得到新的血红蛋白。以光吸收扫描分析修饰血红素和重组血红蛋白,证明新的重组血红蛋白构建成功。酶活力测定表明,修饰血红素得到的重组血红蛋白的类过氧化物酶活性都比天然血红蛋白的酶活力高,用对羟基联苯修饰血红素得到的重组血红蛋白的酶活提高明显,约是天然血红蛋白的1.6倍。     

聚乙二醇修饰超氧化物歧化酶的初步研究

目的:对玉米超氧化物歧化酶(SOD)进行共价修饰,以提高其稳定性.方法:以分子量为6000Da的活化聚乙二醇(PEG)为修饰剂,对玉米超氧化物歧化酶(SOD)进行共价修饰,并确定最佳反应条件.将天然SOD与PEG-SOD分别进行热稳定性、酸碱稳定性及抗蛋白酶稳定性的比较实验.结果:在最佳反应时间10h、最佳反应温度4℃时PEG与SOD反应获得的PEG-SOD比天然SOD在热、酸、碱及抗酶解三方面的稳定性均有不同程度的提高.结论:玉米超氧化物歧化酶经PEG修饰后稳定性显著提高.     

酶化学修饰的应用潜力

本文评论了酶化学修饰在酶工程中的应用潜力,介绍了这方面的最新进展。事实证明,只要选择的化学修饰剂及修饰方法合适,有可能在较大范围内改变酶的性质,如稳定性和溶解度、酶催化活力和选择性等,从而创造天然酶所不具备的优良特性,扩大酶的应用范围。     

功能化离子液体化学修饰猪胰脂肪酶的催化性能

选取氯化1-羧甲基-3-甲基咪唑、氯化1-羧甲基-3-乙基咪唑、氯化1-羧甲基-3-丁基咪唑3种离子液体对猪胰脂肪酶(PPL)进行化学修饰,得到3种修饰的脂肪酶分别命名为PPL-M、PPL-E、PPL-B。以三乙酸甘油酯水解为模型反应,考察离子液体修饰前后PPL的活力、热稳定性、耐有机溶剂性等酶学性质,并通过紫外光谱研究修饰对PPL空间结构的影响。结果表明:修饰后PPL的活力明显提高,对温度和pH的敏感度降低。修饰酶的热稳定性明显提高,在高浓度的甲醇及N,N-二甲基甲酰胺(DMF)中仍能保持游离酶活力的100%。修饰后酶的特征吸收峰发生红移,吸收强度增强,修饰后酶的微环境发生了改变。     

Hydrogen peroxide damages the zinc-binding site of zinc-deficient Cu,Zn superoxide dismutase

Mutations in Cu,Zn superoxide dismutase (Cu,Zn SOD) account for approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a late-onset neurodegenerative disease affecting motor neurons. These mutations decrease protein stability and lower zinc affinity. Zinc-deficient SOD (Cu,E SOD) has altered redox activities and is toxic to motor neurons in vitro. Using bovine SOD, we studied the effects of hydrogen peroxide (H(2)O(2)) on Cu,E SOD and Cu,Zn SOD. Hydrogen peroxide treatment of Cu,E SOD inactivated zinc binding activity six times faster than superoxide dismutase activity, whereas inactivation of dismutase activity occurred at the same rate for both Cu,Zn SOD and Cu,E SOD. Zinc binding by Cu,E SOD was also damaged by simultaneous generation of superoxide and hydrogen peroxide by xanthine oxidase plus xanthine. Although urate, xanthine, and ascorbate can protect superoxide dismutase activity of Cu,Zn SOD from inactivation, they were not effective at protecting Cu,E SOD. Hydrogen peroxide induced subtle changes in the tertiary structure but not the secondary structure of Cu,E SOD as detected by near and far UV circular dichroism. Our results suggest that low levels of hydrogen peroxide could potentially enhance the toxicity of zinc deficient SOD to motor neurons in ALS by rendering zinc loss from SOD irreversible.     

Structural analyses of various Cu2+, Zn2+-superoxide dismutases by differential scanning calorimetry and Raman spectroscopy

The thermal denaturation profile of the Cu2+, Zn2+ metalloenzyme, bovine superoxide dismutase, consists of two primary components, the major component denatures irreversibly at Tm = 104 degrees C with a total enthalpy (delta Hcal) of 7.30 cal/g. Reduction of Cu(II) to Cu(I) with potassium ferrocyanide lowers Tm to 96 degrees C and delta Hcal to 6.96 cal/g. The apo-form of bovine superoxide dismutase (both Cu and Zn removed) denatures at 60 degrees C with an enthalpy only one-half that of the holo-form. The reduced thermal stability, which indicates a greater ability to change conformation, may explain the previously observed much greater membrane binding of the apo-enzyme. Reconstitution with Zn2+, Cu2+, or Zn2+ and Cu2+ raises Tm to 80, 89, or 102 degrees C, respectively, with corresponding increases in the enthalpy. Thus, the metal ions considerably stabilize the enzyme and must somewhat affect conformation. The effect of Cu2+ alone is greater than that of Zn2+, although both are needed for full stability. Raman spectroscopy indicates little difference in secondary structure between the apo- and holo-forms, implying that the increased stability due to metal binding is not caused by an extreme structural reorganization. The value of Tm of canine and yeast superoxide dismutase is also lowered by reduction of Cu(II). The reduced form of the yeast enzyme denatures irreversibly, as do all forms of the bovine and canine enzymes, but the oxidized form is unique in that it denatures reversibly. Thus, the copper ion must be oxidized for renaturation and appears to act as a nucleation site.     

A facilitated electron transfer of copper--zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode

The direct electrochemical redox reaction of bovine erythrocyte copper--zinc superoxide dismutase (Cu(2)Zn(2)SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65 +/- 3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k(s) is 1.2 +/- 0.2 s(-1) and the anodic (alpha(a)) and cathodic (alpha(c)) transfer coefficients are 0.39 +/- 0.02 and 0.61 +/- 0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu(2)Zn(2)SOD), its Cu- or Zn-free derivatives (E(2)Zn(2)SOD and Cu(2)E(2)SOD, E designates an empty site) and the SOD reconstituted from E(2)Zn(2)SOD and Cu(2+). The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.     

重组人超氧化物歧化酶化学修饰的初步研究

在高效表达重组人铜锌超氧化物歧化酶(rh Cu/Zn SOD),并纯化得到比活大于4000单位的 rh Cu/Zn SOD 纯品的基础上,采用活化酯法将聚乙二醇(PEG)与 rhCu/Zn SOD 交联,获得分子量约6万的 PEG-SOD 交联物.经 PEG 修饰的酶稳定性增强,表现为对酸、碱和热的耐受力均较未交联酶高.修饰酶的生物半衰期为15h,是天然酶的90倍,酶活性保留80%以上.还实验观察了修饰剂用量与修饰酶保留活性之间的关系.     

煤矿区耐镉青霉菌的分离鉴定

[目的]分离鉴定煤矸石中耐Cd2+菌株.[方法]用菌落形态和18S rRNA序列分析鉴定菌株,研究菌株的重金属耐性和在酸性煤矸石浸出液的生长能力,分析其抗氧化酶活性对重金属复合污染的响应.[结果]BJKD4菌株为青霉属(Penicillium sp.)菌,能耐29 mmol/L的Cd2+,不同重金属对BJKD4的毒性大小依次为:Cu2+＞Ni2+＞Cd2+＞pb2+或Zn2+＞Mn2+.正交试验表明BJKD4菌株能在不同浓度重金属Cd、Zn、Ni和Mn等复合污染条件下生长,SOD活性在重金属复合污染时升高,CAT活性变化依重金属的种类和浓度不同而不同;此外,BJKD4能在含有煤矸石酸性浸出液的培养基中生长,并提高其pH.[结论]BJKD4菌株能耐多种重金属,具有阻止煤矸石山淋溶液酸化的应用潜力.抗氧化酶在减缓重金属诱导的氧化胁迫中起重要作用.     

Association between dietary fat and antioxidant status of Tunisian type 2 diabetic patients

BACKGROUND: The antioxidant enzymes: superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase (GSH-Px) provide a defense against the damage of cells by reactive oxygen species, which increased in diabetic state. It was demonstrated that dietary treatment could improve the antioxidant status in patients with type 2 diabetes mellitus. This study was undertaken to determine if erythrocyte Cu/Zn SOD and GSH-Px activities correlate with dietary nutrients in 35 selected type 2 diabetic patients (21 women and 14 men) without diabetic complications. RESULTS: We found that erythrocyte Cu/Zn SOD was diminished in patients with poor controlled diabetes and GSH-Px activity was significantly decreased in obese compared with non-obese type 2 diabetic patients (1.07+/-0.87 and 2.36+/-1.99 U/ml, respectively; P=0.024). Both erythrocyte Cu/Zn SOD and GSH-Px activities were positively correlated to erythrocyte omega3-polyunsaturated fatty acids (PUFA). In non-obese diabetic patients, only GSH-Px activity was correlated negatively to the fraction of linoleic acid (18:2omega6) and arachidonic acid (20:4omega6) in erythrocytes phospholipids. CONCLUSIONS: The data of this study reveal that activities of erythrocyte antioxidant enzymes were altered in type 2 diabetic patients. Further studies are needed to determine if diet supplemented with omega3-PUFA is required to improve antioxidant defense system in diabetic state.     

Effects of fluoride on hepatic antioxidant system and transcription of Cu/Zn SOD gene in young pigs

Thirty-two barrows (Duroc x Landrace x Yorkshire) were randomly divided into four groups, each of which included eight pigs. The groups received the same basal diet supplemented with 0, 100, 250 and 400mg/kg fluoride, respectively. The malondialdehyde (MDA) and glutathione (GSH) levels, antioxidant enzymes activities and zinc/copper superoxide dismutase (Cu/Zn SOD) mRNA content in the liver were determined to evaluate the fluoride hepatic intoxication. Results showed the increased lipid peroxides (LPO) level and the reduced GSH content, along with a concomitant decrease in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px). Moreover, the level of hepatic Cu/Zn SOD mRNA was also significantly reduced. We suggest the mechanism of fluoride injuring the liver as follows: fluoride causes a decrease in Cu/Zn SOD mRNA and the reduced activities of antioxidant enzymes, leads to the declined ability of scavenging free radicals with excessive production of LPO, which seriously damages the hepatic structure and function.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Enhanced sensitivity to oxidative stress in Cu,ZnSOD depleted rat erythrocytes.

The effects on red blood cells of superoxide dismutase (Cu,ZnSOD) depletion, induced by feeding Wistar rats with a copper deficient diet, were investigated. SOD depleted red blood cells were more sensitive to peroxidation and to hemolysis than normal cells when exposed to tert-butylhydroperoxide (t-BOOH). Membranes isolated from SOD depleted cells showed a lower content of vitamin E and higher (Na+, K+) and Mg2+ ATPase activities. These results support the view that superoxide dismutase plays an important role in cellular oxidative metabolism.     

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.