Purification and partial characterization of Mn superoxide dismutase from muscle tissue of the shrimp Macrobrachium nipponense

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme had been isolated, purified and partially characterized from muscle tissue of the shrimp Macrobrachium nipponense. The purification was achieved by heat treatment, ammonium sulfate fractionated precipitation and column chromatograph on DEAE-cellulose 32. Some physiological and biochemical characterization of it was tested. The molecular weight of it was about 21.7 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had an absorption peak of 278 nm in ultraviolet region, and the enzyme remained stable at 25-45 degreesC within 90 min. However, it was rapidly inactivated at higher temperature. Treatment of the enzyme with 1 mM ZnCl2, SDS and 1 mM or 10 mM mercaptoethanol showed some increasing activity. However, the enzyme activity was obviously inhibited by 10 mM CaCl2, CuSO4, ZnCl2 and 1 mM CaCl2 and 10 mM K2Cr2O7. SOD activity did not show significantly variation after incubated with 1 mM CaCl2, EDTA and 10 MM SDS. The enzyme was insensitive to cyanide and contained 1.03 +/- 0.14 atoms of manganese per subunit shown in atomic absorption spectroscopy, which revealed that purified SOD was Mn superoxide dismutase. (C) 2004 Elsevier B.V. All rights reserved.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

Algal biotechnology industries and research activities in China

In old China there were very few people engaged in the study of the algae, but in new China, freshwater and marine algae are studied by over one hundred old and new phycologists. There is now an algal biotechnology industry consisting of an aquaculture industry, producing large amounts of the seaweeds Laminaria, Porphyra, Undaria, Gracilaria, eucheumoids, and the microalgae Dunaliella and Spirulina. There is also a phycocolloid industry, producing algin, agar and carrageenan; an industry producing chemicals and drugs, such as iodine, mannitol, phycocyanin, beta -carotene, PSS (propylene glycol alginate sulfate) and FPS (fucose-containing sulfated polysaccharides) and an industry producing food, feed and fertilizer. The Laminaria cultivation industry produces about 900,000 t dry Laminaria, probably the largest producer in the world and 13,000 t algin, undoubtedly one of the largest algin producer in the world.

The complete amino acid sequence of growth hormone and partial amino acid sequence of prolactin and somatolactin from sea perch (Lateolabrax japonicus)

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) were purified simultaneously under alkaline condition (pH 9.0) from pituitary glands of sea perch (Lateolabrax japonicas) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each step of purification, fractions were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with chum salmon GH. PRL and SL antisera. The yields of sea perch GH, PRL and SL were 4.2, 1.0 and 0.28 mg/g wet tissue, respectively. The molecular weights of 19,200 and 20,370 Da were estimated by SDS-PAGE for sea perch GH and PRL, respectively. Two forms of sea perch SL were found: one (28,400 Da) is probably glycosylated, while the other one (23,200 Da) is believed to be deglycosylated. GH bioactivity was examined by an in vivo assay. Intraperitoneal injection of sea perch GH at a dose of 0.01 and 0.1 mug/g body weight at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The complete sea-perch GH amino acid sequence of 187 residues was determined by sequencing fragments cleaved by chemicals and enzymes. Alignment of sea-perch GH with those of other fish GHs revealed that sea-perch GH is most similar to advanced marine fish, such as tuna, gilthead sea bream, yellowfin porgy, red sea bream, bonito and yellow tail with 98.4, 96.2%, 95.7%, 95.2%, 94.1% and 91% sequence identity, respectively. Sea-perch GH has low identity to Atlantic cod (76.5%), hardtail (73.3%), flounder (68.4%), chum salmon (66.3%), carp (54%) and blue shark (38%). Partial amino-acid sequences of 127 of sea-perch PRL and the N-terminal of 16 amino-acid sequence of sea-perch SL have been determined. The data show that sea-perch PRL has a slightly higher sequence identity with tilapia PRL( 73.2%) than with chum salmon PRL(70%) in this 127 amino-acid sequence. (C) 2001 Elsevier Science B.V. All rights reserved.

Purification and characterization of agarases from a marine bacterium Vibrio sp. F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn2+, Mg2+ or Ca2+ increased AG-a activity, while Mn2+, Cu2+ or Ca2+ increased AG-b activity. However, Ag+, Hg2+, Fe3+, EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.

Identification of archaeon-producing hyperthermophilic alpha-amylase and characterization of the alpha-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca2+ for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7-8 and at temperature close to 35 degrees C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40-45 degrees C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.

Settling abalone veliger larvae in a free-swimming microalgal culture

In the current abalone hatchery in China, insufficient diatoms on vertically placed corrugated pvc plates at later stage often could not support the growth of postlarvae up to the stage that they can feed on live macroalgae. As a result, stripping the spats (35 mm) off by anaesthetization and switching the diet from live diatoms to artificial powdered diet in combination has to be performed in most of the abalone farms. This manipulation normally leads to more than 50% mortality. Here we report the direct use of the unicellular green alga Platymonas helgolandica Kylin var. tsingtaoensis as a potential alga to be used to settle the veliger larvae of the Pacific abalone Haliotis discus hannai and to feed the postlarvae. Settlement rate of 2-day-old veliger larvae in mono culture of P helgolandica could be as high as 92% ( +/- 4.2%) on day 10 in small scale trials, higher than that in the selected benthic diatom strain (53.6% +/- 12.7%) when settled in the water in which bacteria propagation was controlled by treatment of 2 ppm of benzylpenicillinum calcium and streptomycin sulfate. Postlarvae fed solely on P. helgolandica or the selected benthic diatom Navicula-2005-A grew at rates of 40.1 ( +/- 1.9) and 45.8 (+/- 13.4) mu m day(-1), respectively, when raised at 22 degrees C until day 50 postfertilization. P. helgolandica was shown to have distinct diurnal settling rhythm characterized with a peak of settled cells in the middle of the night for cell division and a peak of free-swimming cells in the middle of the day. High density of attached P. helgolandica cells on the inner surface of the culture facility in the night fits the nocturnal feeding behavior of the abalone spats. Judged by the promising larvae settling rate, growth and survival rates of the postlarvae fed with this alga, the free-swimming micro-green alga P. helgolandica constitutes a potential species for settling the veliger larvae and for supporting the growth of postlarvae as well. (c) 2006 Published by Elsevier B.V.

Element enrichment and U-series isotopic characteristics of the hydrothermal sulfides at Jade site in the Okinawa Trough

The geochemical and U-series isotopic characteristics of hydrothermal sulfide samples from the Jade site (127A degrees 04.5'E, 27A degrees 15'N, water depth 1300-1450 m) at Jade site in the Okinawa Trough were analyzed. In the hydrothermal sulfide samples bearing sulfate (samples HOK1 and HOK2), the LREEs are relatively enriched. All the hydrothermal sulfide samples except HOK1 belong to Zn-rich hydrothermal sulfide. In comparison with Zn-rich hydrothermal sulfides from other fields, the contents of Zn, Pb, Ag, Cd, Au and Hg are higher, the contents of Fe, Al, Cr, Co, Ni, Sr, Te, Cs, Ti and U lower, and the Pb-210 radioactivity ratios and Pb-210/Pb ratios very low. In the hydrothermal sulfide mainly composed of sphalerite, the correlations between rare elements Hf and U, and Hf and Mn as well as that between dispersive elements Ga and Zn, are strongly positive; also the contents of Au and Ag are related to Fe-sulfide, because the low temperature promotes enrichment of Au and Ag. Meanwhile, the positive correlations between Fe and Bi and between Zn and Cd are not affected by the change of mineral assemblage. Based on the Pb-210/Pb ratios of hydrothermal sulfide samples (3.99x10(-5)-5.42x10(-5)), their U isotopic composition (U-238 content 1.15-2.53 ppm, U-238 activity 1.07-1.87 dpm/g, U-234 activity 1.15-2.09 dpm/g and U-234/U-238 ratio 1.07-1.14) and their Th-232 and Th-230 contents are at base level, and the chronological age of hydrothermal sulfide at Jade site in the Okinawa Trough is between 200 and 2000 yr.

Authigenic sulfide minerals and their sulfur isotopes in sediments of the northern continental slope of the South China Sea and their implications for methane flux and gas hydrate formation

This is a report of the study of the authigenic sulfide minerals and their sulfur isotopes in a sediment core (NH-1) collected on the northern continental slope of the South China Sea, where other geophysical and geochemical evidence seems to suggest gas hydrate formation in the sediments. The study has led to the findings: (1) the pyrite content in sediments was relatively high and its grain size relatively large compared with that in normal pelagic or hemipelagic sediments; (2) the shallowest depth of the acid volatile sulfide (AVS) content maximum was at 437.5 cm (> 2 mu mol/g), which was deeper than that of the authigenic pyrite content maximum (at 141.5-380.5 cm); (3) delta S-34 of authigenic pyrite was positive (maximum: +15 parts per thousand) at depth interval of 250-380 cm; (4) the positive delta S-34 coincided with pyrite enrichment. Compared with the results obtained from the Black Sea sediments by Jorgensen and coworkers, these observations indicated that at the NH-1 site, the depth of the sulfate-methane interface (SMI) would be or once was at about 437.5-547.5 cm and the relatively shallow SMI depth suggested high upward methane fluxes. This was in good agreement with the results obtained from pore water sulfate gradients and core head-space methane concentrations in sediment cores collected in the area. All available evidence suggested that methane gas hydrate formation may exist or may have existed in the underlying sediments.

评价水产动物生长性能的生理生化指标和分子生物学指标

以奥利亚罗非鱼(Oreochromis aureus)为实验对象，设计了3种不同的摄食类型，分别是鲜活饵料组、饥饿3周后饱食投喂组和人工饲料组。鲜活饵料组投喂冰冻赤子爱胜蚓，利用蚯蚓体内丰富的营养成分和活性物质，以期获得奥利亚罗非鱼良好的生长状况；饥饿后饱食组是指饥饿3周后，以人工饲料饱食投喂2周，用于研究饥饿与补偿生长获得快速生长时血液理化指标的变化情况；人工饲料组作为对照组。纯淡水条件下养殖，水温25±2℃。测定了奥利亚罗非鱼在3种摄食类型饲喂下某些血液生理生化指标变化的情况，并将指标变化情况与增重率做相关性分析，试图找出能够反映奥利亚罗非鱼生长性能的血液生理生化指标。 研究结果表明，奥利亚罗非鱼在饥饿3周后获得了补偿生长，补偿生长时的增重率和特定生长率显著高于人工饲料组(P<0.05)，高于鲜活饵料组，但差别不显著；相关性分析研究表明血清总蛋白、胆固醇、四碘甲状腺原氨酸（T4）与增重率极显著相关(P<0.01)，血红蛋白显著相关(P<0.05)，红细胞、白细胞、碱性磷酸酶高度相关(相关系数为0.580、0.551和0.557)，因此，建议血清总蛋白、胆固醇和血红蛋白可作为能够反映罗非鱼生长性能的新指标。 根据序列设计引物，PCR反应条件：变性温度：95 ℃，3 min；退火温度：57℃，20 sec；延伸温度：72℃，5 min，共36个循环，从牙鲆、黑鲪和鲈鱼中克隆出胰岛素样生长因子（IGF-Ⅰ）部分序列，首次证实了IGF-Ⅰ在3种海水鱼中的存在。 利用蛋氨酸与ZnSO4•7H2O，在pH 5.5、80℃下，反应1小时，采用蛋氨酸与硫酸锌2：1的配料比，合成出了产物蛋氨酸螯合锌，蛋氨酸螯合锌外观白色，粉状，室温下微溶于水，不溶于乙醇，并用原子吸收光谱法测定其含锌量为15%，螯合率为88.2%。

迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析

迟缓爱德华氏菌是危害水产养殖业发展的重要病原菌之一，因而其免疫防治研究具有重要意义。论文分析了9种具有保护潜能的迟缓爱德华氏菌蛋白，经过牙鲆免疫保护实验，筛选出EseD和Et18两种有显著性保护效应的抗原。为了提高其保护效应，论文使用基因工程技术将这两种抗原融合到一起，构建重组融合蛋白EEH。结果表明，融合蛋白EEH保护效应较EseD和Et18分别免疫时有所提高。ELISA和Western blotting 结果显示，三种蛋白都能诱导牙鲆产生特异抗体。这些研究为开发迟缓爱德华氏菌疫苗提供了理论基础。 论文克隆分析了迟缓爱德华氏菌AcrAB耐药系统，采用定点突变确定了acrAB、acrR的启动子序列和AcrR在acrAB启动子的结合位点。启动子分析显示，AcrR对acrAB启动子有300倍抑制效应, 对acrR启动子有3倍抑制效应。定点突变显示，K39和R45对AcrR功能具重要性；缺失突变表明，N端205个氨基酸残基是其功能必需。实验筛选出Acriflavine、Ethidium Bromide、Methyl Viologen、Sodium Dodecyl Sulfate等四种AcrR诱导物。分析AcrR过量表达菌株结果显示，其耐药性、生长状况和毒力水平较阴性对照组降低。这些研究加深了我们对迟缓爱德华氏菌耐药机制及其与毒力关系的了解。

硫酸盐还原菌和极化电位对海洋结构用钢在海泥中应力腐蚀开裂敏感性的影响

海洋设施长期处于恶劣的腐蚀环境中，如不加以防护，一旦发生应力腐蚀开裂（SCC），损失就会极为惨重。海底泥土区环境十分重要，因为管线和平台桩腿等都埋在海底泥中。海底泥中硫酸盐还原菌（SRB）十分活跃，而且为了防止腐蚀，海泥中的设施无一例外地采取了阴极保护，相当于设施处在长期稳定的充氢状态。因此非常有必要研究海泥中的活性SRB和极化电位对海洋结构用钢在海泥中的氢渗透行为和SCC敏感性造成的影响，弄清SCC发生和发展的过程以便采取相应的措施减缓或防止SCC。 本文通过慢应变速率拉伸实验（SSRT）、电化学阻抗谱（EIS）技术、动电位扫描极化曲线测定实验和氢渗透实验等研究了海泥中SRB和极化电位对16Mn钢和管线钢X56（API X56）的SCC敏感性造成的影响。 从渤海海泥中富集得到SRB菌种，并做出了SRB在海泥中的生长曲线；在荧光显微镜下观察SRB为弧状，可以归为脱硫弧菌属，为革兰氏阴性菌；海泥中活性SRB数量与硫电位等主要腐蚀环境因子具有一定的对应关系。 SSRT结果表明，施加阴极极化电位可以使试样断裂脆性特征明显，SCC敏感性增大；海泥中活性SRB浓度越高，断裂脆性特征越明显，SCC敏感性越大。在含SRB海泥中或阴极极化电位条件下，两种钢都容易发生SCC，氢脆（HIC）起主要作用。 随着浸泡天数的增加，试样在灭菌海泥中的Rp一直增大；在含SRB海泥中Rp先增大，又变小，并呈现出显著的Warburg阻抗特征；在灭菌海泥中，两种试样在阳极电位范围内无SCC敏感区，而在阴极电位范围内有明显的SCC敏感区；在含SRB海泥中，在阳极电位范围和阴极电位范围内均有SCC敏感区；SRB代谢产物既有阳极去极化作用，又有阴极去极化作用，能使腐蚀电流密度增加。 活性SRB的存在能够促进试样在海泥中的氢渗透；在实海工程应用中，两种钢在含SRB海泥中的氢渗透电流密度大约是在不含SRB海泥中的3~4倍。阴极极化电位能够促进试样在灭菌海泥中的氢渗透。在含SRB海泥中对试样施加阴极极化电位，氢渗透电流密度大于不加阴极极化电位时的氢渗透电流密度，也大于在不含SRB的海泥中的氢渗透电流密度。

硫酸盐还原菌对海洋用钢腐蚀行为的影响及其控制

本文研究了海水和海泥环境中硫酸盐还原菌（SRB）对海洋用钢腐蚀行为的影响及控制，探讨了SRB影响下的腐蚀机制，腐蚀产物的形成及转化过程，并研究了含有SRB的海泥环境中阴极保护对钢腐蚀的影响。 从我国青岛胶州湾海底泥中富集培养出SRB，进行分子生物学分析确定了研究菌种为肠状菌属，并以荧光显微镜和透射电镜（TEM）观察了SRB的形貌特征。 以失重法、电偶腐蚀、交流阻抗（EIS）、电子探针（EPMA）、TEM等手段研究了海洋用钢在含有活性SRB的海泥和海水环境中，从最初的细菌附着到代谢产物导致腐蚀产物从氧化物到硫化物的转化，腐蚀产物的形貌及成分确定，对腐蚀由抑制到加速的过程。此外从钢基体与腐蚀产物界面角度对SRB点蚀的形成和扩展，以及单晶氧化铁立方体在SRB菌液中的生物矿化进行了探讨。 对埋在含有SRB海泥中的低碳钢的阴极保护的可靠性进行评价，重点研究活性细菌存在下不同阴极保护电位下的交流阻抗行为，并结合失重法测试不同电位下的腐蚀速度、MPN法细菌计数以得出保护电位、腐蚀速度以及细菌活性之间的关系。为达到有效的保护，-950mV (CSE)甚至更低的保护电位是需要的。较高保护电位下，细菌的生长活性与稳定性低于低电位。

硫酸盐还原菌对海洋腐蚀行为的影响

本文通过使用多参数水质分析仪、循环伏安法、电化学阻抗法等测试手段研究了硫酸盐还原菌生长过程中硫酸盐还原菌数量、环境参数和碳钢腐蚀行为三者之间的关系；建立了一种有效的快速检测硫酸盐还原菌数量的方法并研究了其检测的机制；研究了硫酸盐还原菌对阴极氧还原反应的影响。 研究发现，硫酸盐还原菌生长过程对介质化学状态影响主要为在硫酸盐还原菌的新陈代谢过程作用下，硫离子浓度在增殖期快速增加，并在衰亡期和残余期保持不变，体系的还原性也与硫离子浓度同步变化。该介质中碳钢腐蚀行为与活性硫酸盐还原菌并无直接关系，主要取决于体系氧化还原性能。即在硫酸盐还原菌增殖期增强了阳极过程与腐蚀速度，并在硫酸盐还原菌衰亡期和残余期保持不变。 采用测定银电极的电极电位的方法可以检测体系中硫酸盐还原菌的数量，检测范围为50~10000ml-1，检测时间为2~3分钟。硫酸盐还原菌的吸附及其代谢生成硫离子的过程是导致银电极电位负移的原因。 在氧气饱和的3.5% NaCl溶液中，玻碳电极的阴极氧还原反应包括氧气一电子还原为超氧离子、氧气二电子还原为过氧化氢以及过氧化氢二电子还原为水分子三个步骤。硫酸盐还原菌催化了第一步和第二步反应中间产物超氧离子和过氧化氢的分解，从而使得阴极氧还原反应电流加大。