高产壳聚糖酶菌株的筛选和发酵产酶条件研究

为探讨专一性地降解甲壳质或壳聚糖的甲壳质酶或壳聚糖酶 ,从采集的土样中分离到 4株产壳聚糖酶能力较强的菌株 ,经摇瓶复筛 ,菌株YJ0 2产酶能力最强。对其发酵产酶条件的研究结果表明其产酶最适培养基组分为 (% ,w/v) :粉末壳聚糖 2 .0 ,葡萄糖 0 .1,NH4NO3 1.0 ,酵母提取物 0 .5 ,K2 HPO40 .0 7,KH2 PO40 .0 3 ,NaCl0 .5 ,MgSO4·7H2 O 0 .0 5 ,起始 pH 6.0。最适产酶培养条件是 :5 0 0mL三角烧瓶装瓶量为 15 0mL ,2 .0 % (v/v)接种量 ,3 0℃ ,15 0r/min培养 72h。在最适产酶培养条件下 ,72h时菌株YJ0 2发酵液中壳聚糖酶活力可达到 17.0 4U/mL。     

产壳聚糖酶菌株选育及培养条件优化

以假单胞菌Y8．0为出发菌株,分别通过亚硝基胍(NTG),Co^60,UV(紫外)诱变,采用透明圈法筛选,获得了产壳聚糖酶较好的突变菌株假单胞菌Y8,其所产酶活力达到3．0U／mL,酶活力提高近6倍,并具有较好的遗传稳定性。3种诱变方法中,UV诱变效果最好。对假单胞菌Y8产壳聚糖酶培养条件进行研究,得到1个比较优化的培养条件为：pH值6．5,温度32℃,壳聚糖3g／L,酵母膏0．3％,培养时间3d。不同金属离子及不同诱导物对酶合成的影响表明：Fe^2 ,Mg^2 激活作用显著。而Zn^2 ,Mn^2 则具有一定的抑制作用,壳聚糖和氨基葡萄糖对壳聚糖酶的产生都有较好的诱导作用。     

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。     

吸附交联法和包埋法固定壳聚糖酶的比较研究

采用吸附交联和包埋法对假单胞菌(Pseudomonas)CUY8产壳聚糖酶进行固定,并对游离壳聚糖酶和2种固定化酶的特性进行比较。游离壳聚糖酶的最佳pH值为5.0,最佳温度为55℃;采用吸附交联方法所获得的壳聚糖酶的最佳pH值为4.0,最佳温度65℃;采用包埋法获得的壳聚糖酶的最佳pH值为5.5,最佳温度为35℃。壳聚糖游离酶,吸附壳聚糖酶和交联壳聚糖酶的米氏常数依次分别为1.92 mg/mL,14.71 mg/mL和5.91 mg/mL;半衰期为18 d,45 d和37 d。实验结果显示,采用吸附交联法所得的壳聚糖酶的半衰期是最长的。     

产壳聚糖酶菌株发酵条件优化及壳聚糖酶的分离纯化研究

从土壤中筛选得到一株产壳聚糖酶菌株P003,对其进行了发酵条件优化.结果表明其最适发酵培养条件为:(NH4)2SO42.0%,粉末壳聚糖1.0%,葡萄糖0.1%,K2HPO40.07%,KH2PO40.03%,NaCl 0.5%,MgSO4·7H2O 0.05%,酵母提取物0.3%,调pH至5.0;2.0%接种量,500 mL三角瓶装液量为150 mL,32℃下150 r/min摇床培养96 h,在此条件下菌株发酵液的壳聚糖酶活力达108 u/mL.采用硫酸铵分级盐析、离子交换层析和凝胶过滤层析等方法,从菌株的发酵液中分离出了分子质量为30.5 ku的壳聚糖酶.     

不结球白菜叶片壳聚糖酶的理化特性研究

通过不结球白菜叶片壳聚糖酶对壳聚糖的降解,研究了温度、pH、时间及金属离子等对此壳聚糖酶的影响。结果表明,壳聚糖酶的最适温度为45℃,最适pH为5.5,酶促反应速度在6 h内达到最大值;Mn2 和Cu2 对壳聚糖酶促反应分别起到促进和抑制的作用。经薄板层析分析证实,壳聚糖酶降解壳聚糖为低聚物。     

壳聚糖酶高产菌株的筛选和发酵条件的研究

从采集的土样中分离到1株产壳聚糖酶能力较强的菌株OU01,并对OU01进行了16S rDNA序列、形态及生理生化鉴定分析。16S rDNA序列分析表明该菌属于微杆菌属;该菌的形态特征与生理生化鉴定结果表明该菌与Microbacteriumsp.的相似性最高;因此将其初步鉴定为微杆菌属(Microbacterium)。同时对该菌的发酵条件进行了初步研究:该菌的最适培养基组成为(g/L):chitosan 10,(NH4)2SO420,MgSO4.7H2O 1.3,K2HPO4.3H2O 1.4,Glucose 1,Yeast extract 3,NaCl 5,起始pH=6.30。最适发酵温度为30℃,最佳发酵时间为96 h,在上述最优条件下,该菌株产酶达到118 U/mL。Microbacteri-umsp.OU01菌株为壳聚糖酶的研究和应用提供了新的来源。     

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。     

壳寡糖和壳聚糖酶水解特性的薄层色谱分析

建立了聚合度2～10的壳寡糖的薄层层析分析(TLC)方法,在异丙醇∶水∶氨水=60∶30∶4的展开体系中,各壳寡糖可以得到很好的分离,各单体间比移值(Rf)差异明显,无拖尾现象。通过对比TLC法与高效液相色谱(HPLC)法分析壳寡糖单体的效果,得出TLC法具有快速、有效、方便的优点,可以作为定性分析壳寡糖单体纯度的方法。采用本实验室制备的壳聚糖酶对3种不同脱乙酰度的壳聚糖和DP2～6的壳寡糖进行水解,采用TLC法对水解产物进行分析。实验结果表明,随着壳聚糖脱乙酰度的降低,其酶水解产物的糖链延长。该壳聚糖酶不能水解DP2～4的壳寡糖,对DP5～6的壳寡糖水解速率缓慢。通过对该壳聚糖酶作用特点分析,表明该酶可作用于GlcN-GlcN糖苷键,而不能水解Glc-NAc-GlcNAc糖苷键。     

壳聚糖酶基因在解脂耶氏酵母中的表达及重组酶性质研究

为构建高效表达壳聚糖酶工程菌株,以Microbacteriumsp.基因组为模板扩增壳聚糖酶基因,目的基因与表达载体pINA1317连接构建重组质粒,重组质粒NotⅠ酶切线性化后转化解脂耶氏酵母Y.liPolytica Po1h。所得结果为PCR扩增得到约1000bp的特异性片段,序列分析表明含有壳聚糖酶全长基因,开放阅读框801bp,编码266个氨基酸残基。转化子酶活力为10.4U/mL,是原菌株酶活力的3.15倍。重组蛋白经DEAE-Sepharose FF分离纯化,达到电泳纯,SDS-PAGE显示单一条带,分子量约41kDa。重组酶反应最适温度为45℃,最适pH为5.6,Km和Vmax分别是0.926mg/mL和6.15U/mL。质谱分析酶解产物主要为三糖和五糖。实现了壳聚糖酶在解脂耶氏酵母中的分泌表达,为壳聚糖酶的工业化生产奠定了理论基础。     

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。     

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.     

Estimation of Sediment Properties during Benthic Impact Experiments

Sediment properties, such as water content and density, have been used to estimate the dry and wet weights, as well as the volume of sediment recovered and discharged, during benthic impact experiments conducted in the Pacific and Indian Oceans. The estimates show that the weights of dry (355-1332 t) and wet sediment (1651-4888 t) recovered during the experiments, are not only a function of the total duration and distance covered during the experiment, but also depend on water content and density of the sediment. Estimation of dry sediment and its volumetric ratio in the discharge, are key parameters for calculating the discharged volume. The estimated volume of wet sediment recovered (1427-4049 m 3 ) as well as discharged (2693-6951 m 3 ) during the experiments, provide important inputs to evaluate the sediment resettlement and migration. Using these estimates, the average depth of excavation on the seafloor, can also been calculated. However, the stages of sediment recovery and discharge are expressed differently in some of the experiments. In order to standardize these, different stages have been identified, and definitions of certain terms have been suggested, for use in the future. The methods of calculating different properties, as well as weight and volume of discharged sediment are described in the paper for use in other applications concerning deep-sea discharges.     

A Tidal Flow Model for the Gulf of Kachchh, India

A vertically integrated model has been used to study the tidal circulation and currents in the Gulf of Kachchh along the west coast of India. The model is fully nonlinear and uses a semiexplicit finite difference scheme to solve the basic hydrodynamic equations on a staggered grid. The model is forced by prescribing the tides along the open boundary of the model domain. The flow is simulated both with and without the presence of the proposed tidal barrage across the Hansthal Creek in the Gulf of Kachchh. The results show a considerable change in the behavior of the tidal flow in the presence of the barrage.     

利用中国GF-3卫星SAR和HY-2A卫星微波散射计的首次台风联合定量观测

<正>The typhoon,as a mature tropical cyclone that develops in the western part of the North Pacific Ocean with high wind speed and heavy rainfall,is one of the most lethal and costly of natural disasters for the densely populated countries of East Asia.It can be easily detected by space-borne sensors operated at microwave,visible or infrared bands(Liu et al.,2014).Synthetic Aperture Radar(SAR)is     

离子交换—原子吸收光谱法测定海水中痕量铜、铅、锌、镉、铁、锰

本文介绍了用国产D401型螯合树脂分离富集海水中铜、铅、锌、镉、铁、锰等痕量元素,并用原子吸收光谱仪测定其含量的方法。讨论了各元素的分离条件选择及干扰元素的影响,并与溶剂萃取法的结果作了比较。各元素检测的定量下限为:铜0.5μg/L、铅0.1μg/L、锌1.0μg/L、镉0.01μg/L、铁2.0μg/L、锰2.0μg/L。方法精密度在4—8％之间,回收率为90—102％。     

Biogeochemistry of Mangrove Soil Organic Matter: a Comparison BetweenRhizophoraandAvicenniaSoils in South-eastern Brazil

Soil core samples fromRhizophora mangleL. andAvicennia schauerianaStapf & Leech forests from south-eastern Brazil were analysed for their total organic matter content and their sugars, amino acid and amino sugars composition. Organic carbon and nitrogen contents were higher inAvicenniathan inRhizophorasoils. The contribution of sugars and amino acids to the total organic carbon pool was constant with depth inRhizophorasoils whereas inAvicenniasoils it increased. Spectral distribution of sugars and amino acids showed a dominance of Ca-affine monomers, particularly acidic amino acids, and the sugar arabinose. Biogeochemical indicators derived from ratios of individual sugar and amino acid monomers confirm previous studies which showed that organic matter in both soils is mainly of mangrove origin. The results further suggest accumulation of organic matter inRhizophorasoils and a continuing degradation of organic matter inAvicenniasoils. The latter may thus release more nutrients to adjacent ecosystems thanRhizophorasoils.     

A review of orange roughy Hoplostethus atlanticus fisheries,estimation methods,biology and stock structure

Orange roughy Hoplostethus atlanticus are unusual fish. They form dense aggregations that have fuelled lucrative fisheries at great depths (600–1 400 m), especially off Namibia, New Zealand and Australia. They are thought to be very long-lived (>100 years, maturity at 22 – 40 years), and to have exceptionally low natural mortality (M = 0.045–0.064 year^?1) and slow growth rates (K = 0.055–0.070 year^?1). In addition, they spawn large eggs and have low fecundity. These factors combine to make orange roughy highly susceptible to overfishing; most stocks are below 30% of pristine levels. Assessments are obtained from indices of catch rate and trawl, acoustic and egg surveys. Acoustic estimates are the most direct, but are confounded by the species' low target strength (?50 to ?53 dB)–attributable to the wax-filled swim bladder. Extracellular wax esters are stored in abundance and comprise mostly mono-unsaturated fatty acids, with low concentrations of the ω-3 fatty acid family. This unusual composition (resultant from the species' diet) ensures neutral buoyancy. Stock separation has been inferred mainly from biological studies, but genetic studies have also found differences among stocks within New Zealand and Australia. Deep-water habitat may be damaged by trawling operations and may take many years to recover, so in some quarters there is a call for a portion of suitable habitat to be set aside for preservation. Although Namibian orange roughy are shallower, smaller and younger than those in other stocks, the Namibian fishery sustained high catches for only a few years before quotas were reduced, from 12 000 to 1 875 tons. Three management lessons are suggested for developing orange roughy fisheries based on the Namibian experience: (1) imposition of catch limits during exploratory fishing; (2) starting the acoustic surveys earlier in the fishery, if possible; (3) greater reliance on trends in catch rate until a survey series has been established.