弧菌DS32褐藻胶裂解酶基因vralg1的异源表达和酶学性质研究

对从福建省东山湾沉积物样品中筛选到的菌株Vibrio sp. DS32的褐藻胶裂解酶基因vralg1进行克隆和异源表达,并对其酶学性质进行评估。以DS32基因组为模板,克隆褐藻胶裂解酶基因vralg1,构建了pET-vralg1重组表达载体,并在大肠杆菌中实现了异源表达,对重组酶VRALG1的酶学性质、底物特异性和完全降解产物等进行了测定。结果表明：重组酶VRALG1最适温度为35℃,在5～50℃范围内相对酶活力达到80%以上,最适pH为6.5～7.5,在pH为6.0～9.0范围内保温1 h后相对酶活力在90%以上;重组酶VRALG1最大反应速率为5.919 mmol/(L·min),米氏常数为3.712 mmol/L,最适条件下比活力为5.874 U/mg; K⁺、Cs⁺、Na⁺、咪唑和乙醇对酶活性影响较小,5 mmol/L或50 mg/mL浓度下相对酶活力保持90%以上,EDTA对酶的抑制作用明显,1 mmol/L浓度下可使酶完全失活;重组酶VRALG1对海藻酸钠和聚古罗糖醛酸具有较高的降解活性,TLC分析显示产物主...     

一株源自海绵的α-葡萄糖苷酶抑制活性菌的鉴定、培养条件优化及活性物质的分离

为从海洋微生物中获取天然α-葡萄糖苷酶抑制活性(α-glucosidase inhibiting,α-GI)化合物,对一株前期研究发现具有α-GI活性的细菌进行鉴定和培养条件优化,并对其代谢产物进行分离,获取和鉴定其活性化合物。通过形态学观察和16S r DNA测序鉴定活性菌株HY95为波茨坦短芽孢杆菌(Brevibacillus borstelensis);采用分析单因素和正交试验选取菌株的最佳培养条件为:2.5%(V/V)的接种量,130 r/min的摇床转速,28℃恒温培养60 h。经优化后的MB培养基中:蛋白胨5.00 g/L,酵母粉1.50g/L、氯化钠9.725 g/L,pH 7.5;以生物活性测试为导向,用化学方法(薄层色谱、高效液相色谱)对其中的活性组分进行分离纯化,并经核磁共振氢谱分析确定得到一个α-GI活性化合为环(苯丙氨酸-酪氨酸),其对α-葡萄糖苷酶的抑制率为53.72%±4.92%。为α-GI活性化合物的筛选提供了一个极有开发前景的途径和来源。     

海水养殖环境中拟除虫菊酯类农药降解菌的分离鉴定及降解特性研究

从近岸海水养殖环境中分离获得一株能高效降解氯氰菊酯(Cypermethrin,CYP)和溴氰菊酯(Deltamethrin,DEL)的菌株HS-10,经生理生化和16S rDNA分析,初步鉴定其为假交替单胞菌(Pseudoalteromonas sp.)。不同降解条件下的实验结果表明,菌株HS-10在pH 7.0、温度28°C的环境中具有较好的生长和降解效率,在该条件下,对初始浓度为100mg/L的CYP和DEL的降解效率分别为75.6%和90.9%。进一步采用发光细菌和核磁共振(NMR)方法对菌株HS-10的降解产物进行分析,结果表明,CYP和DEL主要通过微生物降解消除,其降解产物的毒性显著降低。同时,考察了菌株HS-10对氯氰菊酯、溴氰菊酯、联苯菊酯(BIF)、氟氯氰菊酯(CYF)和氰戊菊酯(FEN)5种拟除虫菊酯类农药(浓度分别为100mg/L)的降解效率,结果表明,对5种农药的降解率均达到50%以上,本研究获得的菌株HS-10可用于海水养殖环境中拟除虫菊酯类农药残留污染的生物修复。     

绿潮藻浒苔微生物高效降解及生物乙醇制备

从绿潮藻暴发海域的底泥和腐烂浒苔(Ulva prolifera)中,筛选出一株可高效降解浒苔纤维素的微生物菌株(F菌株),通过刚果红染色实验以及真菌的ITS分析,鉴定为曲霉属真菌。以浒苔为诱导培养基制备F菌株的粗酶液,测得其滤纸纤维素酶活为34.79 U/m L。在发酵实验中,当酶解条件为6%的粗酶液添加量、反应温度为38℃、反应时间为60 h、pH为6.8时,浒苔纤维素降解效果最好,酶解液发酵乙醇产量最高达到28.98 g/L。     

产新琼四糖的海洋微泡菌属AG1热稳定性β-琼胶酶的过量表达与鉴定

从微泡菌属AG1（Microbulbifer sp. AG1）克隆得到1302 bp大小的琼胶酶基因,该基因编码产物为一个成熟蛋白（413个氨基酸残基）外加一个信号肽（20个残基）。将不含信号肽片段的琼胶酶在E. coli BL21 （DE3）中进行了异源表达和纯化。使用琼脂糖作为底物,该重组琼胶酶的最适反应温度和pH分别为60℃和7.5。该重组酶表现出优良的热稳定性,在50℃和60℃下处理1 h,重组琼胶酶仍能分别保持67%和19%的残余酶活力。除了SDS,重组琼胶酶对于其他测试的抑制剂、去垢剂和尿素变性剂有着较好的抗性。利用薄层色谱和以对硝基苯-α/β-D-吡喃半乳糖苷为底物的酶活力分析结果表明,该重组琼胶酶为β型琼胶酶,它水解琼脂糖的主要终产物为新琼四糖,而且不同聚合度的酶解产物具有抗氧化活性。     

深海真菌Aspergillus puniceus A2次级代谢产物及其药物开发潜力

本研究采用硅胶柱层析、高效液相色谱等方法对深海真菌Aspergillus puniceus A2发酵产物进行了化学成分分离纯化,通过核磁共振等现代波谱学方法,同时结合相关文献进行数据分析,鉴定化合物的结构。从A. puniceus A2中分离鉴定出6个呫吨酮并双呋喃环类化合物,化学结构分别确定为Austocystin I（1）、Austocystin G（2）、6 methoxyl Austocystin A（3）、Austocystin F（4）、F02ZA 1593B2（5）、Austocystin A（6）,化合物1~6均为首次从该真菌中分离得到。通过细胞毒性和抗炎活性评价发现化合物1、2、4和6在20 μmol/L浓度时,对肝星状细胞LX2显示出毒性作用,提示其可能有抗肝纤维化的作用;化合物2、3、5和6在20 μmol/L浓度时,能显著抑制LPS诱导小鼠单核巨噬细胞RAW264.7细胞分泌NO,显示出一定的抗炎活性。     

镉胁迫条件下大弹涂鱼(Boleophthalmus pectinirostris)外周血微核标记及肝脏过氧化物酶标记的变化

采用静态毒性实验方法,研究重金属镉胁迫条件下大弹涂鱼外周血微核率和肝脏过氧化物酶活性的变化。结果表明,在0.05mg/L Cd2 浓度组,10d时检测到微核率极显著升高;0.5mg/L和5mg/L组5d时就可检测到微核率极显著升高,到10d时微核率达到最高值;10d时转入清洁海水后,3个组在15d再次出现一个微核率的最高值。大弹涂鱼肝脏过氧化物酶标记包括谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽转硫酶(GST)。3个组的GSH-Px酶活性和5mg/L浓度组GST酶活性在12h后显示出极显著变化,3个组GST酶活性在24h检测到极显著差异;在转入清洁海水5d时,3个组GSH-Px酶活性和对照组相比可检测到极显著差异,而3个组的GST酶活性均无显著差异。研究结果表明,大弹涂鱼外周血微核标记和肝脏过氧化物酶标记能够灵敏地指示水环境中的镉污染;大弹涂鱼外周血微核标记和肝脏内过氧化物酶标记具有一定的互补作用。     

耗氧速率(OUR)测量方法的实验研究

目前多采用分批实验法和呼吸测量仪测量耗氧速率,而耗氧速率的测量对于研究活性污泥数学模型中的动力学参数及废水特性鉴定具有重要意义。本文根据OUR测量方法的基本原理设计了1种简易的OUR测量装置,为检验测量装置的有效性,设计了相关实验进行验证。在内源呼吸实验中,测定了所用活性污泥的内源呼吸速率是5.1mgO2/L·h,污泥的衰减系数是0.0137h-1,比文献值略高。基质降解实验对人工配水(葡萄糖和淀粉)的耗氧速率进行了测定,实验结果显示有3个显著不同的耗氧速率,分别为35mg/L·h,13mg/L·h,6mg/L·h,它们各代表葡萄糖降解、淀粉降解和内源呼吸的速率。内源呼吸速率高于前1个实验是由于基质降解过程中污泥量增加所致。并计算出异养菌产率系数Kd为0.63mgVSS/mgCOD,Kd略低于文献值。实验结果证明,该装置能很好的实现测量目的。     

水体Hg^2＋对中华绒螯蟹（Eriocheir sinensis）血淋巴抗氧化酶活力和丙二醛含量的影响

采用单因子实验设计方法,进行了水体汞（Hg^2＋）在不同暴露时间对中华绒螯蟹血淋巴超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPX）和过氧化氢酶（CAT）活力以及脂质过氧化产物丙二醛（MDA）含量的影响研究。结果表明,较低浓度Hg^2＋（0．01和0．05mg／L）处理组中华绒螯蟹血淋巴SOD、GPX、CAT活力在...     

孔雀石绿及其代谢物无色孔雀石绿在鲫鱼肌肉中的代谢规律

研究了孔雀石绿及其主要代谢物无色孔雀石绿在鲫鱼肌肉组织内的残留消除趋势。对鲫鱼以200 ng/mL浓度孔雀石绿进行药浴1 h,取肌肉采用高效液相色谱法测定孔雀石绿及代谢物无色孔雀石绿的残留状况。结果表明,孔雀石绿代谢相对较快。代谢产物无色孔雀石绿残留时间较长,可以作为检测的标志物。     

东印度洋分离的一株蓝杆藻的形态学,超显微结构以及系统发生分析

One strain of unicellular greenish algae embedded by mucilage was successfully isolated from equatorial area in the Indian Ocean. Microscopic observation, ultrastructure features and genetic identification confirmed that the strain was closely related to Cyanothece sp., which was a cyanobacteria species with great ecological significance.Cells were solitary with oval or bacilliform shapes. Diameters of this strain were relatively small, ranging from 2.5 to 6.5 μm on average. Ultrastructure of cells was simple. Thylakoids were arranged parietal and keritomized content were observed in the thylakoid region. Various electron-transparent granules with low electron-dense region as well as cyanophycin or glycogen granules-like organelle and carbonxysomes were also observed. For pigment composition, the dominant pigments were chlorophyll a, β-Carotene, Zeaxanthin and an unknown pigment, contributing 23.8%, 26.1%, 14.7% and 15.7% to total pigments respectively. The phylogenetic analysis of16 S rRNA gene and nif H gene confirmed that Strain EIO409 was closely related with Cyanothece sp..     

Effect of Mercury Chloride on the Chlorophyl <Emphasis Type="Italic">a</Emphasis> and Pheophytin Content in Marine Microalgae: Measuring the Flow of Autotrophic Phytoplankton Using Sediment Traps Data

The present work aimed to study the influence of mercuric chloride (HgCl₂) on pigment content (chlorophyll a and pheophytin) in sediment traps on the basis of experimental data. The marine microalgal cultures of Conticribra weissflogii (Mediophyceae), Tetraselmis viridis (Chlorodendrophyceae), Amphidinium carterae (Dinophyceae), Nannochloropsis sp. (Eustigmatophyceae) were used. The obtained results revealed that the influence of mercury on pigment content as well as on photosynthetic activity was different in different species. The considerable decrease in phytopigments content was observed in Amphidinium carterae and Conticribra weissflogii, while the proportion of pheophytin increased. The pigment content of Tetraselmis viridis was not different from the control line, while for Nannochloropsis sp. the increase in pigment concentration was noticed. The complete immediate inhibition of photosynthetic activity was observed in Conticribra weissflogii, Tetraselmis viridis, Amphidinium carterae, while Nannochloropsis sp. responded only after 24 h of exposition. The pigment degradation rate was almost the same for diatoms and dinoflagellates. The equation for estimating the phytopigments content in sediment traps, that takes into consideration the exposition time, is proposed.     

微泡菌QZHA1褐藻胶裂解酶MAAL1的酶学性质研究

为探究Microbulbifer sp. QZHA1褐藻胶裂解(Escherichia coli)酶MAAL1的酶学性质,将褐藻胶裂解酶基因maal1构建至pET-28a表达载体并利用大肠杆菌BL21(DE3)进行异源表达。研究发现：重组酶MAAL1与来源于Microbulbifer sp. ALW1菌株的褐藻胶裂解酶(WP＿23625014.1)同源性最高,为93.69%,且与PL7家族蛋白聚为一支;重组酶MAAL1最适温度为35℃,最适pH为7.5,在pH为5.5～10.5范围内保存24 h仍能保持60%以上的酶活力;MAAL1具备良好的耐有机溶剂特性,在测试的9种有机溶剂中,除异丙醇外,其他有机溶剂在添加量达到30%(体积分数)后,酶活力依然保持在59%以上;重组酶MAAL1最适条件下酶活力为4.3 U/mg,米氏常数(K_m)值为1.08 mg/mL,最大反应速率(V_max)为4.75 mg/(mL·min),催化常数(K_cat)值为4.52 s^-1;重组酶MAAL1对聚β-D-甘露糖醛酸(p...     

西太平洋深海海水中乙醛降解菌多样性及群落结构研究

As a major aldehyde pollutant widely existing in industry and our daily life, acetaldehyde is more and more harmful to human health. As characteristic habitat niche, bacteria from deep sea environments are abundant and distinctive in heredity, physiology and ecological functions. Thus, the development of acetaldehyde-degrading bacteria from deep sea provides a new method to harness acetaldehyde pollutant. Firstly, in this study,acetaldehyde-degrading bacteria in the deep sea water of the West Pacific Ocean were enriched in situ and in the laboratory respectively, and then the diversity of uncultured bacteria was studied by using 16 S r RNA genes. Then acetaldehyde-degrading strains were isolated from two samples, including enrichment in situ and enrichment in laboratory samples of deep sea water from the West Pacific Ocean using acetaldehyde as the sole carbon source,and then the ability of acetaldehyde degradation was detected. Our results showed that the main uncultured bacteria of two samples with different enrichment approaches were similar, including Proteobacteria,Actinobacteria, Firmicutes, Cyanobacteria, but the structure of bacterial community were significant different.Four subgroups, α, γ, δ and ε, were found in Proteobacteria group. The γ-Proteobacteria was dominant(63.5%clones in laboratory enriched sample, 75% clones in situ enriched sample). The species belonged to γ-Proteobacteria and their proportion was nearly identical between the two enrichment samples, and Vibrio was the predominant genus(45% in laboratory enriched sample, 48.5% in situ enriched sample), followed by Halomonas(9% in situ enriched sample) and Streptococcus(6% in laboratory enriched sample). A total of 12 acetaldehyde-degrading strains were isolated from the two samples, which belonged to Vibrio, Halomonas,Pseudoalteromonas, Pseudomonas and Bacillus of γ-Proteobacteria. Strains ACH-L-5, ACH-L-8 and ACH-S-12,belonging to Vibrio and Halomonas, have strong ability of acetaldehyde degradation, which could tolerate 1.5 g/L acetaldehyde and degrade 350 mg/L acetaldehyde within 24 hours. Our results indicated that bacteria of γ-Proteobacteria may play an important role in carbon cycle of deep sea environments, especial the bacteria belonging to Vibrio and Halomonas and these strains was suggested for their potentials in government of aldehyde pollutants.     

中国南麂列岛产海洋底栖硅藻新种-南麂侧链藻(Pleurosira nanjiensis sp. nov.)的形态与分子系统学研究

本文描述了采自南麂列岛小柴屿岩相潮间带的一海洋底栖硅藻新种,南麂侧链藻（Pleurosira nanjiensis sp.nov.）。本研究使用光学显微镜和扫描电子显微镜对该种进行了形态学观察,并基于SSU rRNA和rbcL基因重建了其系统发育关系。南麂侧链藻具有壳面橄榄形、椭圆形或圆形,壳面隆起,单眼突出于壳表面,2–3个唇状突（rimportulae）,放射排列的线纹等特征,区别于其他侧链藻。     

大型水母沙海蜇暴发的温带贫营养海洋生态系统中细菌和浮游植物的关系

Bacterial abundance, phytoplankton community structure and environmental parameters were investigated to study the relationships between bacteria and phytoplankton during giant jellyfish Nemopilema nomurai blooms in the central Yellow Sea during 2013. N. nomurai appeared in June, increased in August, reached a peak and began to degrade in September 2013. Results showed that phosphate was possible a key nutrient for both phytoplankton and bacteria in June, but it changed to nitrate in August and September. Phytoplankton composition significantly changed that pico-phytoplankton relative biomass significantly increased, whereas other size phytoplankton significantly decreased during jellyfish bloom. In June, a significantly positive correlation was observed between chlorophyll a concentration and bacterial abundance(r=0.67, P0.001, n=34).During jellyfish outbreak in August, there was no significant correlation between phytoplankton and bacteria(r=0.11, P0.05, n=25), but the relationship(r=0.71, P0.001, n=31) was rebuilt with jellyfish degradation in September. In August, small size phytoplankton occupied the mixed layer in offshore stations, while bacteria almost distributed evenly in vertical. Chlorophyll a concentration significantly increased from(0.42±0.056) μg/L in June to(0.74±0.174) μg/L in August, while bacterial abundance just slightly increased. Additionally, the negative net community production indicated that community respiration was not entirely determined by the local primary productivity in August. These results indicated that jellyfish blooms potentially affect coupling of phytoplankton and bacteria in marine ecosystems.     

东海富营养化封闭海水养殖海域浮游植物分布与控制因子

The distribution of phytoplankton and its correlation with environmental factors were studied monthly during August 2012 to July 2013 in the Yantian Bay. A total of 147 taxa of phytoplankton were identified, and the average abundance was in the range of 0.57×10~4 to 7.73×10~4 cell/L. A total of 19 species dominated the phytoplankton assemblages, and several species that are widely reported to be responsible for microalgae blooms were the absolutely dominant species, such as Skeletonema costatum, Navicula sp., Thalassionema nitzschioides,Pleurosigma sp., and Licmophora abbreviata. The monthly variabilities in phytoplankton abundance could be explained by water temperature, dissolved oxygen, salinity, dissolved inorganic nitrogen(DIN), and suspended solids. The results of a redundancy analysis showed that p H and nutrients, including DIN and silicate(SiO_4), were the most important environmental factors controlling phytoplankton assemblages in specific months. It was found that nutrients and pH levels that were mainly influenced by mariculture played a vital role in influencing the variation of phytoplankton assemblages in the Yantian Bay. Thus, a reduction of mariculture activities would be an effective way to control microalgae blooms in an enclosed and intensively eutrophic bay.     

中国东南沿海小头虫科(环节动物:多毛类)一新种

中蚓虫是小头虫科较为常见的属,中国海区的样品过去常被鉴定为加洲中蚓虫Mediomastus californiensis Hartman。本研究中,来自东南沿海四个不同海区的118条中蚓虫样品被鉴定为一个新的种类,在此给出具体描述。这个新种具有如下特征：（1）第1至9刚节表皮双环型,刚毛束位于体节中间位置,而后的刚节多环型,刚毛束位于体节后半部;（2）后腹区具有疣足突起;（3）前4个胸刚节仅具双羽型翅毛状刚毛,其它体节仅具巾钩刚毛;（4）胸区巾钩刚毛的柄长且直,巾长为宽的3至5倍;（5）腹区巾钩刚毛不同于胸区,较短且具收缩部;（6）甲基绿染色模式独特。这个研究表明了中国海区的中蚓虫可能被误鉴定为加洲中蚓虫,而这个新种M.chinensis sp.nov.在东南沿海广为分布。     

中国海膨胀巴夫藻(定鞭藻门,巴夫藻纲)的首次纪录

2015年5月,我们从天津近海分离出一株黄绿色具鞭毛的单细胞藻。这珠藻细胞形态不规则,从圆形到细长型都有出现。绝大多数细胞具有很强的运动能力,一些细胞也可以形成静止的群体。结合形态学、超显微结构观察以及分子生物学分析,我们将这株藻鉴定为定鞭藻门,巴夫藻纲的膨胀巴夫藻。在此之前,中国近海只有绿色巴夫藻的记录,绿色巴夫藻对于水产养殖起到了很重要的作用。巴夫藻纲的其他物种尚无中国海记录。这是膨胀巴夫藻在中国海的首次记录。