产碱杆菌DN25中降氰酶的分离纯化及生化特性

以一株可降氰的产碱杆菌DN25为酶来源,通过超滤、30 mg/mL硫酸鱼精蛋白沉淀、30%～70%硫酸铵盐析和Phenyl-Toyopearl 650M疏水层析等步骤,获得比活力为44 U/mg的纯化酶制剂.在确定酶浓度、反应时间等氰降解活力测定条件后开展酶学性质研究,试图为将来氰降解代谢机理的深入研究和菌株的基因工程改造提供理论基础.研究结果表明,此纯化酶催化氰化物水解的最适pH值为8.0,最适温度为30℃.该酶在pH 7.0～8.0区域稳定,而在pH>9时会很快失活;在30℃保存10 h,酶活力保持稳定,高于60℃,酶快速失活.加入甘氨酸稳定剂,在60℃下保存20 min酶活仍可保留19.6%.酶促反应动力学符合米氏双曲线方程,测得米氏常数Km为3.11 mmol/L,最大反应速率Vmax为0.23 mmolL-1min-1.     

一株能产生聚乙烯醇降解酶的委内瑞拉链霉菌

以聚乙烯醇(PVA)为唯一碳源,从土壤中筛选到1株能产生胞外PVA降解酶的放线菌GY1.根据扩增出的该菌株的16SrDNA全序列在GenBank中的比较结果,结合生理生化实验、细胞化学成分及菌落形态分析,确定该菌为委内瑞拉链霉菌(Stretopmycesvenezuelae).GY1菌株以PVA为唯一碳源时,产生的PVA降解酶活性达到120UL-1,是以葡萄糖或可溶性淀粉为唯一碳源时的10倍.当在PVA培养基中添加1gL-1至3gL-1的葡萄糖时,该菌株细胞量和PVA降解酶总酶活随着葡萄糖添加量的增加而增加,最大细胞量是未添加葡萄糖时的2.4倍,最高总酶活是未添加葡萄糖时的2.6倍,而单位质量细胞产生PVA降解酶的能力提高不大.但当添加的葡萄糖浓度从3gL-1增至10gL-1时,总酶活随着细胞量的增加出现下降趋势,同时单位质量细胞产生PVA降解酶的能力也大大降低.在相同PVA聚合度下,高醇解度PVA比低醇解度PVA更有利于GY1菌株的生长及产生PVA降解酶.图5表1参16     

纳豆激酶原核表达载体的构建及其活性鉴定

利用引物搭桥的方法,经PCR扩增,获得了纳豆激酶成熟肽基因(NK),从而构建了大肠杆菌表达质粒NK/pTWIN1,NK/PET32a及NK/PML-c2x,经分别转化宿主菌ER2566,BL21和ER2566,获得了转纳豆激酶基因重组菌.结果表明,NK/pTWIN1-ER2566和NK/PET32a-BL21的纳豆激酶蛋白表达量较高.本研究选用NK/pTWIN1-ER2566作为表达菌株,对其进行发酵表达.SDS-PAGE显示,目的蛋白约占菌体总蛋白的36%,该蛋白是以包涵体形式存在的.包涵体经收集、蛋白变性及复性,并经过SP Sepharose分离纯化,得到了纯化的纳豆激酶蛋白,琼脂糖-纤维蛋白平板法测出1mg纳豆激酶干粉的溶栓活性相当于600u尿激酶.本文从基因工程角度研究纳豆激酶基因的克隆、表达及纯化,为利用基因工程菌生产纳豆激酶奠定了基础.图5表1参11     

Phosphate solubilizing actinomycetes in the estuarine environment: an inventory.

Sediment samples were collected from different stations of the Vellar estuary for isolation of total actinomycetes and phosphate solubilizing actinomycetes. Phosphatase activity in the sediments was also investigated Consistently a higher number of actinomycetes, phosphate solubilizing actinomycetes and phosphatase activity were recorded from the clay sediments than the sandy sediments at all the stations. In all, 7 strains showed positive phosphatase activity. Among them, one strain PS-3 exhibited good activity and was further investigated for optimum phosphorus solubilization at different pH (6, 6.5, 7, 7.5 and 8) and incubation (1st day to 20th day) periods. The solubilizing activity was maximum at the pH 7 and an incubation period of 13 days was required for an appreciable quantity of phosphorus to be leached into the medium. Based on the chemotaxonomical and conventional methods of identification, the strain PS-3 has been tentatively identified as Streptomyces galbus. The present study indicates that phosphatase enzyme and S. galbus along with other actinomycetes species would play a major role in solubilizing the phosphate in the estuarine ecosystem and increasing the soluble phosphate concentration thereby enhancing the productivity     

降解菌djl-6多菌灵水解酶的提取及性质

以往对多菌灵降解菌Rhodococcus qingshengii sp.nov.djl-6的降解途径研究显示,该菌株首先通过多菌灵水解酶将多菌灵水解成二氨基苯并咪唑,从而对多菌灵进行脱毒.为开发酶制剂并有效应用于环境中残留污染物多菌灵的降解,比较了不同提取方法(高压细胞破碎、超声波破碎和添加溶菌酶破碎)对多菌灵水解酶提取效率的影响,并对其酶学特性进行了初步研究.结果表明,djl-6菌株在LB培养基中培养72~84 h,生长量和产酶量均达到最大值.采用超声波破碎提取酶的效率较高(蛋白浓度为7.92 mg/mL),但酶活损失较大(比酶活只有1.2 U/μg protein).多菌灵水解酶属于一种胞内组成型酶.该酶水解多菌灵的最适pH值为7.0,最适温度为30℃,Zn2+和K+对酶活有一定的抑制作用.     

The production and secreton of digestive enzymes in the purple seastar Pisaster ochraceus

The purple seastar Pisaster ochraceus contains clearly measurable protease and amylase activity. Centrifuged supernatants of pyloric caeca homogenates undergo a spontaneous threefold increase in protease activity when incubated under toluene for 50 h at 25°C. Amylase activity remains nearly constant over this period. Bovine trypsin at a 1 to 100 ratio (trypsin to supernatant protein) induces a twofold increase in protease activity over that of the control supernatant while having virtually no effect on amylase activity over the control. The data indicate a specific interaction of trypsin with a protease zymogen rather than a conspecific hydrolysis of membrane components or vesicles by trypsin. Two percent Triton X-100 used as a diluent in place of distilled, deionized water causes a sevenfold increase in protease activity and a twofold increase in amylase activity in pyloric caeca supernatants. The use of Triton as a diluent in the preparation of a stomach-tissue supernatant allows quantitative measurement of both protease and amylase activity in that tissue.     

猪精液酸性N-乙酰-β-D-氨基葡萄糖苷酶的纯化及性质水

以杜洛克猪精液为材料,采用硫酸铵分级沉淀、DEAE-32阴离子交换柱层析和Sephadex G-100分子筛柱层析纯化,获得纯化倍数为27.64、比活力为1 773.25 U mg~(-1)的酸性N-乙酰-β-D-氨基葡萄糖苷酶纯酶制剂.经SDS-聚丙烯酰胺凝胶电泳法测定,纯酶两种亚基相对分子质量分别为129.13×10~3和62.24×10~3.对其酶学性质的研究结果表明,酶的等电点为5.10,最适pH值为5.5,最适温度为60℃.酶在pH 3.6~9.2、温度10℃～55 ℃的范围内较稳定.以对硝基苯-N-乙酰-β-D-氨罐葡萄糖苷(pNP-NAG)为底物,测得米氏常数K_m为0.455 mmol L~(-1),最大反应速度V_m为17.34μmol L~(-1)min~(-1),活化能为41.70 kJ mol~(-1).图8表1参15     

Joint effects of Penta-BDE and heavy metals on Daphnia magna survival, its antioxidant enzyme activities and lipid peroxidation

The joint toxicity of Penta-BDE (Pe-BDE) and heavy metals including cadmium and copper on Daphnia magna (D. magna) was evaluated on the basis of determining the 48 h survival, antioxidative enzyme responses, and lipid peroxidation. The response was classified as additive, greater than additive, or less than additive by comparing the measured ??toxic units, TU?? with one. Based on the survival of D. magna, less-than-additive interactions were found in most of mixtures treatments. This may be attributed to the different toxicity mechanism between Pe-BDE and metals. Cu and Cd played a greater role in toxicity than what Pe-BDE did. As for the superoxide dismutase (SOD) and catalase (CAT) activity, most response was less than additive. For the glutathione S-transferases (GST) activity, most of the greater-thanadditive responses were found in the Cu plus Pe-BDE treatments, but the additive responses occurred in Cd plus Pe-BDE treatments and binary metal treatments. For lipid peroxide levels, which were measured as malondialdehyde (MDA) levels, less-than-additive response occurred in the 50% Cd plus 50% Cu and ternary mixture treatments. Results suggested that Pe-BDE, Cd, and Cu could induce different patterns of antioxidant enzyme responses, such as antioxidant/prooxidant responses, depending on their capability to produce reactive oxygen species and antioxidant enzymes to detoxify them.     

Size exclusion chromatography for the removal of pigments from extracellular ligninolytic enzyme extracts from decayed wheat straw

Solid-state fermentation of wheat straw was carried out by a native white rot basidiomycete Daedaleopsis flavida strain 5A. Extract prepared from the 12-day decayed wheat straw contained extracellular ligninolytic enzymes like manganese peroxidase (MnP), manganese-independent peroxidase (MIP), lignin peroxidase (LiP) and laccase along with straw-degraded products and pigments. Sephacryl S-200 size exclusion chromatography in 16/100 column was used for the separation of these ligninolytic enzymes and straw-degraded products and pigments. Recovery of pigment-free ligninolytic enzyme activities as protein was 40% of the total proteins loaded and specific LiP activity increased 34 fold after size exclusion chromatography. Thus accurate estimation of LiP by veratryl alcohol oxidation assay was possible only after the removal of interfering pigments. The reproducibility of size exclusion chromatography is adjudged satisfactory from the consistent results obtained after seven repetitive uses of matrices.     

中国红豆杉内生放线菌En-1的分离、鉴定及其次生代谢物的研究

采用选择性分离方法对药用植物红豆杉Taxus chinensis嫩枝和叶进行内生菌分离,得到一株放线菌En-1,经鉴定为链霉菌Streptomyces sp.;并对该菌进行液体培养条件优化、次生代谢物初筛及其抗菌活性的研究。通过添加宿主植物浸出物至En-1液体培养中以考察宿主对后者生长的影响,结果表明红豆杉叶浸出物能促进En-1的体外生长;而且En-1次生代谢物对5株供试菌中的黑曲霉具有抑制活性。通过En-1菌培养基优化并联合波谱法检测其发酵产物,显示8#配方（淀粉25 g.L-1,KNO3 1 g.L-1,K2HPO4.3H2O 0.5 g.L-1,NaCl 0.5 g.L-1,FeSO4.7H2O 0.01 g.L-1,MgSO4.7H2O 0.5 g.L-1）培养液中,En-1所产的次生代谢物最具结构多样性;同时8#配方也有利于该菌产生抗菌活性产物。本研究提示红豆杉内生放线菌可作为寻求药物先导化合物的资源菌。     

Cesium inhibition of motor activity in comparison to Lithium and Rubidium †

The biochemical and toxicological significance of cesium is scarcely understood, and could be evaluated in comparison with lithium widely used as a psychotropic drug. Two male Wistar rat groups of 200–220 g are administered independently, lithium, sodium, rubidium and cesium chloride, in doses of 3mEq/Kg/day (0.024 Eq/L drinking water) during 29 days. Motor activity was measured after the injection of 70 mg pargyline/Kg animal i.p. as inhibitor of MAO A + B with an activimeter of Tedeschy type. Accumulative movements per minute are presented in function of time. Total brain proteins, alkaline and acid phosphatases and blood parameters, haematocrit, haemoglobin and erythrocytes, were determined.

The maximal increase of motor activity was seen in rats treated with RbCl 2 h after the pargyline administration and the diminution was Rb>Li>Cs. Cesium induced a decrease of the total serum protein concentration from 6.39 ± 0.1 to 5.8±0.5mg/100ml serum in controls. Acid and alkaline phosphatase were decreased in cesium treated rats. The three determined blood parameters, haematocrit, haemoglobin and erythrocytes, show also a decrement with cesium treatment compared to the control ones.     

一种嗜热细菌来源角质酶的分离纯化及酶学性质

通过跟踪发酵液中pNPB水解酶活性,对角质诱导的Thermobifida fusca 口发酵液进行分离纯化.采用活性炭脱色、硫铵沉淀、Phenyl HP疏水色谱、DEAE sephamse阴离子交换色谱等方法,分离纯化得到电泳纯PNPB水解酶.该酶水解角质可得到角质单体,是一种角质酶.SDS-PAGE电泳结果显示,角质酶表观分子量约为29×10~3.该酶的最适温度为60℃.在40℃和60℃下均具有良好的热稳定性.最适pH为8.0,pH稳定范围为6.0～9.0.该角质酶的生化性质适合在纺织工业中应用.图8表2参17     

Influence of endosulfan and monocrotophos exposure on the activity of NADPH cytochrome C reductase (NCCR) of Labeo rohita (Ham)

The response of NADPH cytochrome C reductase (NCCR) activity in liver of Labeo rohita fish exposed to the pesticides, 0.25 microgl(-1) endosulfan and 2 mg/l monocrotophos was studied. In terms of specific enzyme activity (mU/mg protein) a significant level of NCCR was observed in the liver tissues of Labeo rohita exposed to the pesticides, when compared to the control fish (2.460 mU/mg protein). Increase of NCCR activity was more in the liver of the fish exposed to monocrotophos (4.595 mU/mg protein) than those exposed to endosulfan (2.850 mU/mg protein). The results demonstrate that the pesticides, endosulfan and monocrotophos, interfere with NADPH dependent monoxygenase mechanism and are effective inducers of NADPH cytochrome C reductase. The activity of NCCR in the liver tissue of Labeo rohita may serve as a useful tool for monitoring aquatic pollution.     

β-甘露聚糖酶基因在枯草芽孢杆菌中的克隆及表达

从Bacillus subtilis JNA 3-10中克隆出β-甘露聚糖酶基因成熟肽链编码序列manA1和含信号肽的β-甘露聚糖酶基因manA2,在B.subtilis 168中克隆表达,分别筛选获得高效分泌表达β-甘露聚糖酶的重组菌株BPM1001(pMA5-manA1/B.subtilis 168)和BPM1002(pMA5-manA2/B.subtilis 168),结果表明菌株BPM1002总酶活力是菌株BPM1001的9.65倍,是原始菌株的13.1倍.在基因manA2下游引入His序列克隆出β-甘露聚糖酶基因manA3,获得枯草芽孢杆菌168重组菌株BPM1003.采用Ni-NTA柱纯化重组菌株BPM1003分泌表达的β-甘露聚糖酶,并研究其酶学性质,该酶促反应的最适pH为6.5,最适温度为65℃,在37℃条件下保存一个月酶活力依然保留有77.8%.5 L发酵罐放大实验结果表明魔芋粉对于产β-甘露聚糖酶具有明显的诱导作用,酶活力最高可达2 748.82 U/mL.图9表3参19     

Post natal antioxidant enzyme activity of rat brain regions during developmental lead exposure

This study reports the effects of low level developmental Pb exposure on specific brain regions like hippocampus, cerebellum and cerebral hemispheres of antioxidant enzyme activities. Wistar dams were exposed to 50 ppm, 100 ppm and 500 ppm of Pb acetate in drinking water during pregnancy and lactation (gestation day 6 through PND 21 (post natal day) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were determined in the hippocampus, cerebellum and cerebral hemispheres of pups during treatment period (PND 7, 14, and 21 days) and also during withdrawl period (PND 35, 45, 60 and 90 days). During treatment period, SOD activity significantly (p < 0.05) decreased in all regions of all the treated groups with maximum decrease in 500 ppm treated group of 21 days, while GSH-Px and GR activities increased with maximum increase in 21 days aged 500 ppm group. During withdrawl period, the activities of all enzymes were significantly (p < 0.05) reversed. Thus the perinatal exposure of dams to variable dosages of low level lead results in characteristic neurochemical alterations in rat brain regions due to impaired antioxidants function.     

一株降解原油链霉菌的分离鉴定与降解特性研究

从山东东营胜利油田附近被石油污染的土壤中分离得到1株高效原油降解菌Z1a-B,依据形态和培养特征,初步鉴定Z1a-B菌株为链霉菌属白孢类群。试验结果表明,该菌株具有较强的溶血和排油活性,说明其产生生物表面活性剂的能力较强。通过气相色谱-质谱联用仪(GC-MS)分析,菌株能基本降解C12~C34的正构烷烃,对烷基苯、菲、甲基菲、萘也具有较强的降解能力。固体培养基配比以麸皮2 g、鸡粪40 g、草炭52.5 g、生石灰1.5g,或麸皮2 g、大米20 g、黄豆粉28 g、生石灰1.5 g较佳,在这2种配比培养基中链霉菌生长快,长势好,产孢子量多。经室内培养试验发现,当土壤中w(原油)=10%时,经Z1a-B菌株处理(35℃,pH 6.5)50 d,土壤原油降解率为67.5%。     

Freeze survival of the cyanobacteria Microcoleus chthonoplastes without cryoprotector

A Microcoleus chthonoplastes strain SC7B9002-1 isolated from microbial mats in tidal channels from San Carlos, Baja California Sur, Mexico was subjected to short- (15 days) and long-term (2 years) conservation assays in liquid nitrogen (-196 degrees C) using cryoprotective agents, such as 5% DMSO, 20% PVP-40, and 20% glycerol. Survival rate, chlorophyll a, protein, and nucleic acids content were observed in each case. Interesting growth and a significant increase in protein content was observed when no cryoprotectant was used during liquid nitrogen immersion. In the absence of a cryoprotectant, M. chthonoplastes lost their typical shape resembled spheroplasts, and recovery cultivation times after freezing were 5 and 25 days (short and long-term, respectively). Recovery from long-term preservation with 5% DMSO took 15 days. PVP and glycerol did not allow recovery of viable cells. The survival of M. chthonoplastes to freezing without cryoprotectant and the adaptive mechanisms that allow surviving under freezing conditions are discussed.     

洞庭湖湿地Cd富集植物蒌蒿(Artemisia selengensis)的耐性生理机制研究

采用盆栽植株和外源Cd胁迫的方法,分别对不同生长期蒌蒿植株根、叶组织中的抗氧化酶活性、可溶性蛋白及MDA含量进行测定,以揭示蒌蒿在Cd胁迫下的抗氧化机理和耐受机制。研究结果表明,Cd胁迫对上述生理指标均有显著影响:≤20mg·kg-1的Cd处理可使幼苗期植株器官中的可溶性蛋白含量增加16.5%～19.1%,100mg·kg-1的Cd含量水平则导致其含量减少近30%,可溶性蛋白含量的变化与植株的生物量积累关系密切;3种酶活性在0～10mg·kg-1的Cd处理下未显示出明显变化,幼苗期植株CAT、POD活性在10～80mg·kg-1的Cd胁迫下增加明显,高于此浓度范围则使酶蛋白受到破坏而失活,中等含量水平(≤40mg·kg-1)的Cd处理经过长时间作用可使植株逐渐适应胁迫环境,胁迫强度较大的Cd处理可显著提高SOD酶的活性;Cd胁迫过程中植株MDA积累量不断增加。3种抗氧化酶活性的增强在蒌蒿植株耐受Cd胁迫方面能起到较好的防御作用。     

Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea musselMytilus galloprovincialis

6-phosphofructo-2-kinase (PFK-2) from the mantle of the sea musselMytilus galloprovincialis Lmk, collected from the Ría de Arosa (NW Spain) in 1990, was purified 550-fold by extraction and sequential affinity chromatography on Affi-gel Blue and ATP-agarose columns. The enzyme was a dimer with a native molecular weight of 100 kilodaltons (KDa) and a subunitM _r of 53 KDa. PFK-2 activity is dependent on the presence of P_i. At physiological P_i concentrations, the enzyme exhibits hyperbolic kinetics with both ATP and Fru-6-P, withK _m values of 0.62 and 0.37 mM respectively. In vivo, PFK-2 activity is limited by the concentration of Fru-6-P which is low in comparison with theK _m for this substrate. Citrate and PEP inhibited PFK-2 activity.