中国明对虾蛋白酶的分离纯化及其酶学性质研究

以中国明对虾(Fenneropenaeus chinensis)肝胰脏为材料,通过硫酸铵沉淀分级分离、Sephadex G-100和DEAE-32柱层析纯化,经PAGE获得蛋白酶,该酶的比活力为96.752U/mg。SDS-PAGE显示一谱带,测得酶亚基分子量为36.3kDa,凝胶层析法测得酶分子量为100.9kDa,推断该蛋白酶由3个相同亚基组成,等电聚焦电泳法测得酶的等电点pI为9.65。以酪蛋白为底物,研究蛋白酶催化反应的动力学参数。结果表明:蛋白酶的最适pH、最适温度、Km和Vmax分别为8.0、65℃、4.578mg/mL和0.273U/min;酶在pH为5.0~10.0范围内较稳定,在20~45℃之间具有较好的热稳定性,在50℃以上热稳定性迅速下降。Mg2+对酶活力没有影响,Ca2+、Ba2+、Cu2+、Mn2+、Co2+、Zn2+和Hg2+对酶有不同程度的抑制作用,其中Hg2+的抑制作用最强,10mmol/L Hg2+可使酶活力完全丧失,而Fe2+对酶有激活作用。EDTA对酶活力没有影响,推断该蛋白酶为非金属蛋白酶。     

南大洋深海嗜冷菌2-5-10-1及其低温脂肪酶的研究

从南大洋普里兹湾的水样中筛选到一株产低温脂肪酶的深海嗜冷菌2-5-10-1,对其生长及产酶情况和酶性质做了初步研究.该菌株的最适生长温度为5℃,此时分泌的胞外脂肪酶最多;添加Tween80,橄榄油可显著促进脂肪酶的产生.该脂肪酶的最适作用温度为35℃,在0～20℃均保持较高的酶活性,在0℃可保持37%的相对酶活性;酶的最适作用的pH值为7.5,在pH6～9的范围内均存在较高酶活性;对热较敏感,在60℃保温15min可丧失50%以上的酶活性.该脂肪酶的催化作用不需要金属离子的参与,Cu2+和Zn2+对酶活有着强烈的抑制作用.     

南极产碱性淀粉酶菌株NJ270的选育及酶学性质

从南极中山站排污口沉积物样品中筛选到一株产碱性淀粉酶的耐冷菌株NJ270,结合形态学观察和16S rDNA序列分析表明该菌属于假单胞菌属(Pseudomonas).发酵试验发现添加淀粉能使产酶量提高8.42倍.采用硫酸铵沉淀、Q Sepharose XL离子交换层析和Sephadex G-75凝胶层析对假单胞菌NJ270淀粉酶进行了纯化,获得电泳纯的淀粉酶,SDS-PAGE检测结果表明该酶大小约为55 kDa.酶学性质研究表明其最适pH值为9.0,最适作用温度为50℃.在低于40℃的条件下具有较高的热稳定性,属于碱性中温淀粉酶.该酶可水解可溶性淀粉生成麦芽五糖.酶活力受多种金属离子和螯合剂的影响,其中Mg2+可使酶活力提高10%,而Fe3+、Co2+、Cu2+、Zn2+、Hg2+、EDTA则能抑制酶活性,抑制率均为60%左右.该酶的性质特征表明其在洗涤剂制造等工业生产中有一定的应用潜力.     

黄鱼(Hexagrammos otakii)脑组织AChE的亲和层析

采用亲和层析法对黄鱼脑组织的乙酰胆碱酯酶进行分离纯化。纯化酶最大比活力为63．136U／mg蛋白，最适pH为8，最适反应温度为35℃。此酶经过SDS-PAGE呈一条带，表明其纯度达电泳纯。对纯化酶进行底物特异性、过量底物抑制效应和抑制剂选择性检验，结果证实该酶为乙酰胆碱酯酶。     

壳聚糖酶的分离纯化及性质研究

以本实验室分离的产壳聚糖酶YJ02菌株出发,制备壳聚糖酶发酵液,经离心、(NH4)2SO4分级盐析、Q-SepharoseFast Flow阴离子交换、Sephacry S-100凝胶过滤分离纯化步骤,SDS-PAGE显示为一条带,壳聚糖酶纯化了28.9倍,收率为47.6%。该酶的性质研究表明,该酶的分子量为66.2KD,最适反应温度为50℃,最适反应pH为6.0;Mn2 对酶活力有明显的促进作用,Cu2 ,Fe3 ,Hg2 对酶活力有抑制作用;小分子有机物EDTA和SDS对酶活力有抑制作用,而IAA和β-巯基乙醇对酶活力稍有促进作用。该壳聚糖酶对高脱乙酰度壳聚糖底物水解作用强,酶解位点可能为NGlc-NGlc,酶最大反应速度Vmax=4.1μmol/min,常数Km为64mmol/L。此壳聚糖酶水解壳聚糖制备的壳寡糖数均分子量为2 400Da。     

海洋弧菌碱性蛋白酶的分离纯化及部分性质研究

采用硫酸铵沉淀、Sephadex- 75 ,Sephadex- 10 0凝胶过滤层析等方法纯化海洋弧菌 (Vibriop acini) X4 B- 7菌株产生的碱性蛋白酶 ,得到电泳纯酶制品 ,并对纯化酶的性质进行了研究。结果显示 :纯酶的分子量为 2 7KD,等电点 p I=8.7,最适反应 p H9.0～ 10 .5 ,最适反应温度 5 0～ 6 0℃。ED-TA对酶活力没有影响 ,高酶浓度可以降低 SDS对酶的抑制作用 ,该酶可用于解聚组蛋白。 DNA琼脂糖凝胶电泳证明 :酶对 DNA酶有降解作用 ,而对 DNA没有降解作用 ,该酶有希望应用于核酸的提取     

产甲壳素酶菌株的发酵条件、酶的分离纯化及酶学性质研究

利用甲壳素为唯一碳源从土壤中筛选得到1株产甲壳素酶活力较高的菌株HD002,初步鉴定其为Massilia属。确定菌株产甲壳素酶的最适培养基组成为(g/L):(NH4)2SO45,K2HPO40.7,KH2PO40.3,MgSO4.7H2O 1,胶体甲壳素10;最适产酶培养条件为:培养基起始pH值6.0,培养时间192 h,发酵液酶活力达1.314 U/mL。采用70%饱和度硫酸铵盐析、DEAE-Sepharose F.F阴离子交换层析、Sephadex G-75凝胶过滤层析对该甲壳素酶进行纯化,SDS-PAGE证明达到电泳纯。该甲壳素酶的分子量为60.2 kDa,最适反应温度为55℃,最适反应pH值为5.0,Cu2+和Fe3+对酶活力有明显的抑制作用,Ca2+和Na+对酶活力有明显的促进作用。     

中国明对虾体壁N-乙酰-β-D-氨基葡萄糖苷酶的分离纯化及酶学性质

为了探讨对虾N-乙酰-β-D-氨基葡萄糖苷酶(EC3.2.1.52,NAGase)的分离纯化及其酶学性质,作者以中国明对虾(Fenneropenaeus chinensis)体壁为材料,通过硫酸铵沉淀分级分离、SephadexG-100柱层析和DEAE-32离子交换柱层析纯化,获得聚丙烯酰胺凝胶电泳纯的N-乙酰-β-D-氨基葡萄糖苷酶酶制剂,纯化酶比活力为3938.56U/mg。通过SDS-PAGE凝胶电泳,测得该酶亚基分子量为48.88 kD。酶的最适pH为6.0,最适温度为45℃;该酶在pH 6.0~9.0区域较稳定,温度稳定性范围是20~35℃,45℃下处理1 h酶活力丧失65.04%。酶水解对硝基苯-N-乙酰-β-D-氨基葡萄糖苷的Km为0.229 mmol/L,Vmax为5.00μmol/(L·min)。进一步研究金属离子对酶活力的影响,结果表明:Li+、Na+和Ba2+对酶没有明显影响,Mg2+、Ca2+、Mn2+、Co2+、Fe3+和Al3+对酶均有不同程度的激活作用,Cu2+、Zn2+和Pb2+对酶呈抑制作用,1.0 mmol/L Hg2+对酶呈激活作用,而10 mmol/L Hg2+使酶活力降低42.37%。     

低温脂肪酶基因在毕赤酵母中的表达研究

将已有的低温脂肪酶基因克隆至表达载体pPICZα上,并转化毕赤酵母X-33。在低温条件下诱导培养6d后,发酵液中可检测到低温脂肪酶活力。重组酵母发酵液加入二硫苏糖醇后单位酶活力达到32U/mL,与未加入该试剂的原始菌株相比提高4倍以上。对重组脂肪酶的酶学性质研究表明,其最适pH为8.0,最适温度为35℃,50℃温浴30min后,仍有30.04%的活性。以不同碳链长度的4-Nitrophenyl Ester为底物检测其底物特异性,结果显示其较适合作用于短链底物。重组酵母能够在BMMY培养基中胞外分泌表达带有His标签的重组脂肪酶,经镍柱纯化后,SDS-PAGE检测可得到大约35kDa的单一蛋白质条带。     

极地产适冷脂肪酶菌株的酶学性质研究

从南北极环境样品中分离到7株产脂肪酶的细菌，经16SrDNA序列分析表明，这些细菌分别属于假交替单胞菌属（Pseudoalteromonas）和嗜冷杆菌属（Psychrobacter）．采用p-NPP法对这7株细菌所产的脂肪酶进行酶学性质研究，结果显示这些酶的最适作用温度在30～40cC、最适作用pH值在7—8之间，在低温下能保持较高的剩余活力，对热敏感，属于适冷脂肪酶．其中假交替单胞菌（Psychrobacter sp．7342）所产脂肪酶具有低温下酶的剩余活力高、有效作用温度和pH范围广、热稳定性较好及对多种金属离子抗性强等特点．该菌株能利用多种单一氮源和碳源产酶，最适产酶温度为25℃．在此基础上进行正交实验分析得到了该菌株的最佳发酵条件为：蛋白胨和淀粉含量各为1．33％，酵母膏含量为0．3％，温度为25℃．     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

Assessing the Validity of Seismo-Acoustic Predictor Equations for Obtaining Seabed and Sub-Surface Sediment Physical Properties

The interdependence between the seismo-acoustic properties of a marine sediment and its geotechnical/physical parameters has been known for many years, and it has been postulated that this should allow the extraction of geotechnical information from seismic data. Though in the literature many correlations have been published for the surficial layer, there is a lack of information for greater sediment depths. In this article, a desktop study on a synthetic seafloor model illustrates how the application of published near-surface prediction equations to subsurface sediments (up to several tens of meters burial depth) can lead to spurious predictions. To test this further, acoustic and geotechnical properties were measured on a number of sediment core samples, some of which were subjected to loading in acoustically-equipped consolidation cells (oedometers) to simulate greater burial depth conditions. For low effective pressures (representing small burial depths extending to around 10 meters subsurface), the general applicability of established relationships was confirmed: the prediction of porosity, bulk density, and mean grain size from acoustic velocity and impedance appears generally possible for the investigated sedimentary environments. As effective pressure increases through, the observed relationships deviate more and more from the established ones for the near-surface area. For the samples tested in this study, in some instances increasing pressure even resulted in decreasing velocities. There are several possible explanations for this abnormal behavior, including the presence of gas, overconsolidation, or bimodal grain size distribution. The results indicate that an appropriate depth correction must be introduced into the published prediction equations in order to obtain reliable estimates of physical sediment properties for greater subsurface depths.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

Interannual Sea Level Variations and Annual Tides in the Northwestern Pacific

Specific properties of the interannual sea level variations and annual tides in the Northwestern Pacific were studied. Several tide stations were monitored. The monthly mean sea level for the year of 1995 was analyzed at each tide station. A seismic event in 1995, some tectonic activity around the subject area, and the Kuroshio (the oceanic western boundary current) may possibly contaminate results which would have occurred from the astronomical annual tide alone.     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.     

Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picophytoplankton: Fixation and Storage Effects

The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics.     

Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans

A set of 27 marine planktonic bacteria isolated from the polar regions was characterized by 16S rDNA sequencing and physiological and biochemical testing. More than half of these bacteria were positive for caseinase, gelatinase and 13-glucosidase, and could utilize glucose, maltose or malic acid as carbon source for cell growth. Twelve isolates expressed nitrate reduction activities. Except for one antarctic isolate BSwlO175 belonging to Actinobacteria phylum, these isolates were classified as γ-Proteobacteria, suggesting that γ-Proteobacteria dominated in cultivable marine bacterioplankton at both poles. Genus Pseudoalteromonas was the predominant group in the Chukchi Sea and the Bering Sea, and genus ShewaneUa dominated in cultivable bacterioplankton in the Prydz Bay. With sequence similarities above 97%, genus Psychrobacter was found at both poles. These 27 isolates were psychrotolerant, and significant 16S rDNA sequence similarities were found not only between arctic and antarctic marine bacteria （ 〉 99% ）, but also between polar marine bacteria and bacteria from other aquatic environments （ ≥ 98.8% ）, including temperate ocean, deep sea, pond and lake, suggesting that in the polar oceans less temperature-sensitive bacteria may be cosmopolitan and have a bipolar, even global, distribution at the species level.