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1.
深海适冷菌SM9913产生的低温蛋白酶 总被引：14，自引：2，他引：14
从1855m深的深海泥样中分离纯化得到200多株分泌蛋白酶的适冷菌，其中3株产低温蛋白酶，本文对其中一株Pseudomonas sp.SM9913(P.SM9913)生长的适冷性和它产生的蛋白酶的适低温特性进行了研究。该菌株能够在0℃正常生长，其最适生长温度为15℃，最高生长温度为35℃。为一株适冷菌。该菌株所产蛋白酶的比合成速率在10℃时最高，催化酪蛋白水解的最适温度为35℃，在0℃仍具有3％的酶活力。最适pH为8．0。该蛋白酶的热稳定性很低，在40℃保温10min即丧失85％的活力，40℃时的半衰期为6min，为一典型的低温酶。抑制剂试验表明，该蛋白酶为金属蛋白酶。 相似文献
2.
采用MSM寡营养培养基从印度尼西亚Pantai cermin,Kalianda和Banyuwedang三个地区的热泉水样、泥样以及沉积物样品中分离获得细菌菌株,通过检测蛋白酶产生透明圈和福林酚蛋白酶活性测定相结合的方法,从中筛选出1株产嗜热蛋白酶的菌株PBI,该菌株经初步鉴定为短杆菌属(Brevibacillus),并对其酶学性质进行了研究。结果表明,菌株PBI产蛋白酶的最适酶活温度为60℃,最适pH值为8.0～9.0,具有较好的热稳定性和pH稳定性,60℃时酶的半衰期为30min,70℃条件下20min仍保持46.1%的酶活,该酶在pH值为5.0～9.0范围的缓冲液中酶活相对稳定。其产嗜热蛋白酶的酶活力最高可达到60.53U/mL,在100℃条件下仍能保留26.37%的相对酶活。Fe2+,Fe3+,Cu2+和Zn2+对嗜热蛋白酶活性具有明显的抑制作用。该嗜热蛋白酶对EDTA敏感,苯甲基磺酰氟(PMSF)、亮抑酶肽(Leupeptin)、苄眯(Benzamidine)和胃蛋白酶抑制剂(Pepstain A)对该嗜热蛋白酶都有一定的抑制作用,说明其酶活性受到丝氨酸、半胱氨酸残基的影响。结果表明,该菌株是1株具有进一步改造利用价值的产蛋白酶菌株。 相似文献
3.
以三疣梭子蟹(Portunus trituberculatus)肝胰腺中丝氨酸蛋白酶为研究对象,对其进行超声波和加热处理,通过紫外光谱、荧光光谱分析和蛋白质变性凝胶电泳分析丝氨酸蛋白酶的变化。研究表明,超声功率和超声时间对丝氨酸蛋白酶的酶活有不同影响。低功率短时间超声处理会使酶活力升高,随着功率和超声时间的增加,酶活力逐渐降低。紫外光谱显示,超声处理使酶液紫外吸收出现蓝移现象,而加热对其影响不大。内源荧光显示,超声和加热处理均对蛋白酶有荧光猝灭作用。SDS-PAGE显示,超声和加热2种处理方法均能引起丝氨酸蛋白酶不同程度的降解。 相似文献
4.
海洋弧菌碱性蛋白酶的分离纯化及部分性质研究 总被引：2，自引：0，他引：2
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 相似文献
5.
南海沉积物细菌胞外蛋白酶在家族水平上的多样性 总被引：2，自引：0，他引：2
海洋产蛋白酶细菌及其分泌的胞外蛋白酶在降解有机氮以推动海洋氮循环进行方面发挥着重要作用，但目前对这二者多样性的认识非常有限。本研究自南海沉积物中筛选得到90株产蛋白酶细菌，并通过N-端氨基酸序列，分析了其胞外蛋白酶在家族水平上的多样性。16S rRNA基因序列分析的结果表明，筛选的产蛋白酶细菌均属于Gammaproteobacteria纲，且大多数属于Alteromonadales目和Vibrionales目的不同属。对其中14株菌株的14个胞外蛋白酶的N-端氨基酸序列分析表明，所有这些蛋白酶属于金属蛋白酶的M4家族或丝氨酸蛋白酶的S8家族。本研究提供了海洋沉积物产蛋白酶细菌在类群及其胞外蛋白酶在类型上的新细节，这将有助于全面了解微生物酶促降解海洋沉积物有机氮的过程和机理。 相似文献
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微生物降解羽毛既可保护环境又能实现废物的资源化利用,具有显著的经济效益和社会效益。本研究从广西钦州港海鸭养殖淤泥筛选获得一株可高效降解羽毛的菌株Gxun-20,通过对该菌株形态特征、生理生化特性和16S rRNA基因序列进行分析,以确定菌株的分类,并对菌株产角蛋白酶的条件及酶学性质进行研究。结果表明：该菌为副短短芽孢杆菌(Brevibacillus parabrevis);菌株最适产酶条件为：温度34 ℃,初始pH 6.5,羽毛含量1.5%(质量分数),发酵48 h,角蛋白酶活性可达277.45 U/mL。菌株Gxun-20所分泌的角蛋白酶最适温度为50 ℃,且热稳定性较好,最适pH为7.5;金属离子Fe3+、Cu2+、Co2+、Fe2+和Mn2+对酶活性有显著抑制作用,而Ca2+、Na+对酶活性有明显促进作用;金属螯合剂乙二胺四乙酸(ethylene diamine tetraacetic acid, EDTA)可显著抑制酶的活性,蛋白酶抑制剂苯甲基磺酰氟(pheny methyl sulfonyl fluoride, PMSF)可完全抑制酶活性,表明该蛋白酶的活性中心含有金属离子和丝氨酸;异丙醇可使酶活性提高至129.34%,酶活性在十二烷基硫酸钠(sodium dodecyl sulfate, SDS)、吐温40溶液中保持稳定;酶的底物特异性结果表明,该酶对羽毛类β-角蛋白及酪蛋白降解能力较好,而对牛血清蛋白、I型胶原及人发类α 角蛋白降解能力较弱。海洋来源的副短短芽孢杆菌Gxun-20及其所产角蛋白酶在废弃羽毛降解方面具有良好的应用价值。 相似文献
7.
利用选择性筛选培养基从所构建的深海沉积物宏基因组文库中筛选得到一株产蛋白酶的克隆（CAPR0002）,对其进行了酶学性质分析．结果表明该酶的最适作用温度为65cc,最适作用pH值为9．0．该蛋白酶具有较好的热稳定性,在40cC以下的温度中可长期保持稳定,在50℃中处理6h后仍能保持60％的活力,在60℃下保温30rain后仍能保持约60％的活力．Ca^2＋、Mg^2＋、sr^2＋、co^2＋对该蛋白酶有明显的促进作用,而且ca^2＋的存在可明显提高该蛋白酶的热稳定性,ca^2＋、sr^2＋、c0^2＋这3种离子均在3．0mmol／dm^3。浓度时具有最高的促进作用,当浓度高于3．0mmol／dm’时,这3种离子对酶活力的促进作用减弱．Hg^2＋、Fe^2＋、cu¨对酶有明显的抑制作用．CAPR02蛋白酶在pH值为7。5～9．5时活力较高,pH值为7。5时可保持约80％的活力,pH值为9．5时保持60％的活力,而在pH值高于9．5的条件下酶活力下降较快,pH值为10．0时活力降为约15％,表明CAPR02属于碱性蛋白酶．丝氨酸蛋白酶抑制剂PMSF、E一64和AEBSF对CAPR02蛋白酶均无抑制作用,显示该酶不属于丝氨酸蛋白酶,而EDTA对酶有明显的抑制作用,表明该酶属于金属蛋白酶． 相似文献
8.
从舟山海域潮间带海泥筛选到产碱性蛋白酶的海洋放线菌,利用Folin-酚法进行酶活测定,选取酶活最高的菌株进行鉴定,绘制系统进化树。再通过紫外线和DES诱变从而得到高产碱性蛋白酶的海洋放线菌菌株,并进行初步的发酵条件研究。结果表明:分离筛选得到2株菌株,在脱脂奶筛选平板上能产生较大的水解透明圈,通过Folin-酚法进行酶活测定,挑选出酶活较高的菌株A20(初始酶活为104.7U/mL),生理生化试验和16S rDNA试验结果显示该菌株为Streptomyces roseus。对菌株A20进行紫外线和硫酸二乙酯(DES)诱变,最终得到高产碱性蛋白酶的菌株,酶活为227.5U/mL,酶活提高117.3%,传代试验显示该菌株具有较好的遗传稳定性。单因素试验显示最适发酵温度为50°C、pH值为9.0、培养时间为72h。 相似文献
9.
《中国海洋大学学报(自然科学版)》2015,(6)
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。 相似文献
10.
本文在太平洋深海沉积物中分离得到一株孔雀石绿降解菌株，鉴定命名为Tenacibaculum sp.HMG1。通过菌株生长实验和高效液相色谱的研究表明， HMG1菌株可以在20 mg/L的孔雀石绿中维持较快的生长速率，并且在12 h内可降解98.8%的孔雀石绿，这证明该菌株具有很高的孔雀石绿耐受能力和降解活性。通过基因组测序在HMG1菌株发现一条过氧化物酶基因可能参与了孔雀石绿的降解，随后利用原核表达获得了相应的重组蛋白。实验表明，该重组过氧化物酶具有极强的活性，可在1000 mg/L的孔雀石绿中发挥降解功能。本文利用液相色谱-质谱联用（LC-MS）技术对孔雀石绿的菌株降解产物和重组酶降解产物进行鉴定，并基于鉴定结果推测了两种降解途径。结果发现两种降解方式存在共同的降解途径。此外，孔雀石绿降解条件的实验结果证明重组过氧化物酶可以在低温（20℃）、复杂的pH值（6.0–9.0）、高盐度（100 mmol/L）、金属离子和EDTA等反应条件下依旧维持很高的孔雀石绿降解活性。以上实验结果表明，HMG1菌株和重组过氧化物酶均在孔雀石绿污染生物修复方面具有很大潜力。 相似文献
11.
Chandralata Raghukumar G. Sheelu P. A. Loka Bharathi Shanta Nair C. Mohandass 《Marine Georesources & Geotechnology》2001,19(1):1-16
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. 相似文献
12.
Arne Schnack-Friedrichsen Angela M. Davis Jim D. Bennell Dei G. Huws 《Marine Georesources & Geotechnology》2001,19(4):221-243
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. 相似文献
13.
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。 相似文献
14.
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. 相似文献
15.
Shigehisa Nakamura 《Marine Geodesy》2000,23(1):55-61
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. 相似文献
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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. 相似文献
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Phylogenetic diversity and phenotypic characterization of cultivable bacterioplankton isolated from polar oceans 总被引：1，自引：0，他引：1
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. 相似文献
20.
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. 相似文献