绿侧花海葵(Anthopleura anjunae)寡肽制备的关键技术与抗前列腺癌作用研究

本文探讨绿侧花海葵(Anthopleura anjunae)抗前列腺癌酶解寡肽制备与工艺优化。以绿侧花海葵肉为原料进行酶解,筛选最佳蛋白酶,通过正交实验优化酶解条件,并通过超滤、阴离子交换层析、G-25凝胶过滤和反相高效液相色谱(RP-HPLC)分离纯化,通过LC-MS和氨基酸序列测定鉴定寡肽的氨基酸序列,并以MTT法检测产物对前列腺癌细胞系DU-145增殖抑制率,以确定活性最强组分,最后通过倒置显微镜观察纯化肽的抗前列腺癌活性。结果表明,碱性蛋白酶为最佳酶种;工艺条件为:最适料液比1︰5、最适p H=11、最适加酶量2000U/g、最适温度35oC、最适酶解时间6h;经高效液相纯化得到由五个氨基酸组成的绿侧花海葵抗前列腺癌寡肽,其氨基酸序列为:Tyr-Val-Pro-Gly-Pro(AAP-H);倒置显微镜结果显示经AAP-H作用24h后的DU-145细胞具有明显的凋亡形态学特征。结论:采用碱性蛋白酶酶解并通过超滤和色谱分离技术能从绿侧花海葵肉中制备抗肿瘤活性肽;且AAP-H对DU-145细胞的增殖抑制作用呈时间与剂量依赖关系,作用后细胞出现了凋亡的形态学特征。因此,AAP-H能明显抑制前列腺癌细胞DU-145增殖,可以诱导其发生凋亡而发挥抗肿瘤作用。     

酶解马氏珠母贝肉制备降血压肽工艺条件优化

以马氏珠母贝（Pinctada martensii）肉为原料,选用6种蛋白酶在各自适宜的条件下进行酶解,采用HPLC法分析酶解产物对血管肾张素转换酶（ACE）的抑制活性（IP）,筛选出碱性蛋白酶为其制备的最佳酶种类,并确定了水解度为21％时,产物具有较好的抑制活性。在此基础上,分别考察了加酶量,底物浓度,酶解温度及pH对水解度的影响。结果表明：当加酶量为0．175g／L,底物浓度为35g／L,温度为45℃,pH为9．5,水解时间240min为酶解最适宜工艺条件。通过SDS-聚丙烯酰胺凝胶电泳测定分子质量分布,表明酶解产物中主要为14．4ku以下的多肽分子。     

酶解糙刺参内脏团蛋白制备抗氧化肽的工艺研究

为了能够有效利用糙刺参内脏蛋白,以水解度为主要指标,对酶解蛋白酶进行了筛选,并系统研究了酶解温度、p H、加酶量和水解时间等单因素对水解度的影响。在此基础上,利用响应面中心组合设计实验,对酶解工艺进行了优化,得到以温度、p H、加酶量及水解时间为因子的二次方程,通过方差分析和验证性实验得出,此二次方程能较好反应海参内脏团蛋白水解度的变化规律,得到最佳水解条件为:温度52.66℃,p H 6.71,加酶量2.5%(质量分数),酶解时间10 h,预测最高水解度为49.58%。最佳酶解条件下所得多肽74%以上分子质量小于500 Da。利用DPPH法测定最佳条件酶解所得多肽的抗氧化活性,3 g/L的抗氧化率达到了24.8%,利用ABTS法测定最佳条件酶解所得多肽的抗氧化活性,3 g/L的抗氧化率达到了0.255μmol/g。为海参内脏蛋白的利用提供了参考。     

动物蛋白酶1058水解ti鱼可溶性蛋白质

研究了动物蛋白酶 1 0 58水解鱼是鱼蛋白时 ,酶量、温度、作用时间对鱼是鱼蛋白质水解率和苦味的影响。结果表明 :酶浓度的提高 ,水解时间的延长 ,均使鱼是鱼蛋白水解率明显提高。最适水解温度为 50℃ ,当温度高于 50℃时 ,水解率逐渐下降 ,蛋白酶开始失活。苦味值随水解率提高而增加。用正交试验的方法 ,经综合分析和试验得出 ,该蛋白酶水解鱼是鱼的最佳条件是酶量与底物之比为 1 .5∶ 1 0 0 0 ;温度为 50℃ ;水解时间为 4h。     

酶促反应制备壳寡糖及壳寡糖分析

用壳聚糖酶降解法对壳聚糖进行降解制备壳低聚糖,研究了温度、pH、底物浓度、脱乙酰度、反应时间对酶促反应的影响。对酶解产物进行HPLC分析,用Bio—Gel P-4柱对酶解产物进行初步分离。结果表明,壳聚糖酶降解壳聚糖的最适温度为45℃,pH为6．0,最适底物质量分数为6％,脱乙酰度越高酶解反应所得产物的数均相对分子质量越小,24h后酶解反应达到平衡,酶解产物壳寡糖水溶性极好。P-4柱分离得到聚合度20以上,10～20,10以下3种壳寡糖。     

海产品中蛋白酶产生菌的选育及产酶条件研究

从海产品虾和蟹中分离出蛋白酶产生菌共37株，其中YM007菌株蛋白酶生产活力较高，为194．6U／mL。以YM007为出发菌株，通过物理和化学方法复合诱变，获得了YM079号菌株。对其产酶条件的研究结果表明：它的最适产酶温度为35℃，最适生长pH为7．0，经过190r／min摇床培养36～40h，其产酶活力为240U／mL，比出发菌株的产酶活力提高了22％。该菌株经发酵的粗酶液最适作用pH为7．0，最适作用温度是45℃，是一种中温型中性蛋白酶。     

响应面法优化酶法制备藻蓝蛋白抗氧化肽的实验研究

为优化碱性蛋白酶酶解藻蓝蛋白制备抗氧化肽的工艺条件,采用响应面分析法,以超氧阴离子自由基清除率为响应值,研究了[E]/[S]、酶解温度和时间对制备抗氧化肽工艺的影响.结果表明,制备藻蓝蛋白肽的最佳酶解条件为:温度为44.9℃、时间为6.4 h和[E]/[S]为3.6%,此时对超氧阴离子自由基清除率达到75.39%.在该条件下制备的藻蓝蛋白酶解产物(0.5 g/L)还原能力的吸光度为1.03,并达到稳定.     

褐藻酸降解酶的制备及其性质研究

通过培养褐藻酸降解菌交替单胞菌(Alteromonas sp．)菌株H-1使其产酶，研究了该酶的性质。结果表明，该菌在25℃培养72h时产酶量最高。褐藻酸酶作用的最适底物质量分数为1％～2％。最适pH值为7．5，最适反应温度为40℃，温度升高酶活力急剧下降。     

一种产血纤维蛋白溶酶菌株的筛选及液体发酵条件的选择

主要进行了产生纤维蛋白溶酶菌株的筛选培养和液体发酵条件的优化等方面的研究工作。结果表明，该菌为一种短杆状菌，革兰氏染色为阴性，具有对血纤维蛋白的产酶依赖性。用此菌作为菌种进行发酵产酶的最适条件为：碳源，木糖１．５％，氮源，血纤维蛋白１．７５％；发酵液初始ＰＨ７．４；接种量５％；发酵时间２４h。     

响应面法优化罗非鱼(Oreochromis niloticus)鱼排蛋白分段酶解工艺

采用生物蛋白酶酶解罗非鱼鱼排蛋白,以水解度(DH)为指标,进行了单酶、复合酶酶解效果的比较分析,并利用响应面分析方法(RSM)对酶解参数进行优化。结果表明,采用风味蛋白酶和木瓜蛋白酶双酶组合分段酶解,固定总酶添加量为3.0%、双酶复合比2:1、酶解时间3h、分段酶解时间比0.6:1,酶解温度56℃,分段酶解pH为7.0和6.2时,酶解效果最佳,罗非鱼鱼排蛋白水解度达到32.49%,酶解产物中游离氨基酸含量达32.84mg/g鱼排,占罗非鱼鱼排总氨基酸含量的25.43%,其中呈味氨基酸含量达18.48%,必需氨基酸含量达67.96%。     

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.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

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.     

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。     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

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.     

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.