Purification and characterisation of vanadium haloperoxidases from the brown alga Pelvetia canaliculata

Two enzymes characterised as iodoperoxidases (PcI and PcII), with vanadium-dependent activity, have been purified from the brown alga Pelvetia canaliculata (L.) Decne et Thur. (Fucaceae, Phaeophyceae), collected in the Northern Portuguese coast, at Viana do Castelo. The relative molecular masses were 166 kDa for PcI and 416 kDa for PcII, as determined by gel filtration. SDS-PAGE shows that PcI has just one band corresponding to a subunit of 66 kDa, while PcII shows four bands (66, 72, 157 and 280 kDa). The following kinetic parameters have been determined from a steady-state analysis of the oxidation of iodide by H2O2: PcI, pHopt = 6.0, KM(I-) = 2.1 mM, KM(H2O2) = 110 microM, Ki(I-) = 127 mM; and PcII, pHopt = 6.5, KM(I-) = 2.4 mM, KM(H2O2) = 20 microM and Ki(I-) = 69 mM. These iodoperoxidases are thermostable, as also observed for vanadium bromo- and chloroperoxidases.     

A Marine Strain of Flavobacteriaceae Utilizes Brown Seaweed Fucoidan

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) with a yield of about 3.8% dry weight. To isolate enzymes that degrade fucoidan, we first screened marine bacteria for their ability to utilize fucoidan, and isolated one strain of Flavobacteriaceae from seawater that could do this. Phylogenetic analysis of the 16S ribosomal DNA sequence suggested that this strain appeared to belong to a new genus, and was tentatively named Fucobacter marina. The strain utilized L-fucose (17%), D-mannose (91%), D-galactose (46%), and D-glucuronic acid (66%) in the fucoidan from K. crassifolia. The strain partially utilized fucoidan from 2 other seaweeds that belong to the order Laminariales, Undaria pinnatifida (10%) and Lessonia nigrescens (48%).     

Antitumor Active Fucoidan from the Brown Seaweed,Umitoranoo (Sargassum thunbergii)

Neutral and acidic polysaccharides and their protein complexes were fractionated and purified from the brown seaweed umitoranoo (Sargassum thunbergii) by fractional extraction, iron-exchange chromatography, and gel filtration. Thirty-one polysaccharide fractions were obtained and tested for antitumor activity in mice with Ehrlich carcinoma transplanted i.p. Two of the fractions, GIV-A ( – 127° and mol. wt., 19,000) and GIV-B ( – 110° and mol. wt., 13,500) had such activity. On the basis of chemical and spectral analyses, these compounds were found to be a fucoidan or L-fucan containing approx. 30% sulfate ester groups per fucose residue, about 10% uronic acid, and less than 2% protein.     

褐藻多糖的分离提取及生理活性研究进展

褐藻多糖是海藻胶、褐藻糖胶和褐藻淀粉的统称,主要存在于褐藻中。褐藻糖胶作为其主要的生理活性物质,主要由L-岩藻糖和硫酸酯基组成,具有抗氧化、抗凝血、防癌抗肿瘤、抗病毒和消炎等活性。综述了褐藻多糖的提取分离方法和褐藻糖胶的生理活性研究进展,以期为褐藻多糖的应用提供参考。     

日粮添加海藻粉对种蛋孵化的影响

目的:研究日粮中添加不同比例的海藻粉对种蛋孵化的影响。方法:在相同饲养条件下,随机抽取600枚种蛋进行孵化实验,每组150枚。其中Ⅰ组为对照组的种蛋,Ⅱ、Ⅲ、Ⅳ组为试验组,分别添加不同水平(1%、3%、5%)的海藻粉的种蛋。结果:结果表明:饲料中添加适量海藻粉可以显著提高种蛋的孵化率和健雏率(P0.05)。结论:以上结果说明,饲料中添加海藻粉可提高种蛋的孵化率和健雏率,且添加5.0%海藻粉孵化效果最佳。     

Polar Constituents of Brown Seaweed Colpomenia peregrina (Sauv.) Hamel from the Black Sea

GC-MS of trimethylsilyl derivatives of the compounds present in the butanolic extract of biomass of brown seaweed Colpomenia peregrina from the Black Sea aided in identification of 24 components, including aliphatic hydroxy and keto and aromatic acids, glycerol, mannitol, floridoside, and monosaccharides. The polysaccharide composition of the biomass was also studied, with high sodium alginate and laminaran contents and a comparatively low level of fucoidan being revealed. The polysaccharides were isolated from the biomass by fractional extraction and purified by precipitation or ion exchange chromatography. The structures of alginic acid and laminaran were deduced from ¹³C NMR spectra and confirmed, in the case of laminaran, by methylation analysis. The sodium alginate was shown to contain more guluronic (G) than mannuronic acid (M) residues, the M/G ratio being 0.48. Laminaran was demonstrated to be a -glucan with 1 3 linkages in its backbone and 1 6 linkages in its branching points, which is characteristic of brown algae. Fucoidan turned out to be a complex heteropolysaccharide containing, in addition to fucose and sulfate, other neutral monosaccharides and uronic acids.     

发菜藻蓝蛋白分离纯化的研究

以发菜为材料,比较了提取液类型和饱和硫酸铵浓度对藻蓝蛋白提取的影响,并对藻蓝蛋白的提取程序和部分特性进行了研究。结果表明:50 mmol/L KP缓冲液(pH值7.2)是合适的提取液,体积分数为40%~50%饱和硫酸铵盐析效果优于其它浓度。经过DEAE-Toyopeal 650 S离子交换层析和SuperdexTM200凝胶过滤层析后,藻蓝蛋白纯度达6.2,最大吸收峰位于615 nm,荧光发射峰位于649 nm,由α和β2个亚基组成,其分子质量分别为18 051.17和19 142.27 Da。因此,发菜藻蓝蛋白分离纯化较为理想的程序为:藻粉→50 mmol/L KP缓冲液(pH值7.2)浸泡→French pressure(1 500 kg/cm2)破碎细胞→40%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S离子交换层析→SuperdexTM200凝胶过滤层析→较纯的藻蓝蛋白。     

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5''-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.     

Isolation and Purification of Prodigiosin from Vibrio psychroerythrus

The red pigment of Vibrio psychroerythrus (formerly marine psychrophile NRC 1004) was identified as prodigiosin by comparison of its mass spectrum, absorption spectrum in the visible range, and chromatographic behavior with prodigiosin isolated from Serratia marcescens. The properties of the V. psychroerythrus pigment were clearly distinguishable from five other prodigiosin-like compounds isolated from three different microorganisms.     

从人脐带血中分离纯化血纤溶酶原

以人血清为原料 ,利用纤溶酶原对L型赖氨酸的高亲和性制备了Lysine -Sepharose4B和Lysine -Agarose ,以亲和层析法从人血浆中提取和纯化血纤溶酶原 (plasminogen ,PGn)。利用聚丙烯酰胺凝胶电泳对其纯度和分子量进行分析 ,结果表明纯化得到的为 92kDa的单一组分的人血纤溶酶原。这种纯化方法的建立为进一步大量制备血管生成抑制素 (angiostatin)奠定了基础。     

微小毛霉凝乳酶的分离纯化研究

研究微小毛霉(HL-1)凝乳酶的分离纯化条件及方法。研究酶的最适浸提温度、酶的浸提pH值和最适浸提时间,探讨离子浓度、加水量对浸提效率的影响,利用高速冷冻离心法、有机溶剂沉淀法,膜分离法和层析法等对粗酶液进行了分离。利用光谱法对纯化样品进行检测。酶的最适浸提温度为30℃;最适pH为6.0;浸提10 h活力最高;1%的氯化钠有利于酶的分离,加水比例为15时有利于提取,在10 000 r/min下离心10min澄清效果最好,95%的酒精沉淀效果最好,利用0.2μm的微滤膜可除去发酵液中的菌体,8 000的超滤膜可拦截凝乳酶蛋白,S300的填料可有效分离凝乳酶,纯度达95%以上。     

Detection and Identification of Fungi Intimately Associated with the Brown Seaweed Fucus serratus

The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.     

Induction of Attachment of the Mussel Perna perna by Natural Products from the Brown Seaweed Stypopodium zonale

Marine invertebrates settle, attach, and/or metamorphose in response to signals from several sources, including seaweeds. In response to the aquaculture challenge of producing constant numbers of juveniles from cultured species, natural inducers have been screened for their ability to improve those processes. However, few chemical inducers of attachment of invertebrates have been identified, and even less of these were secondary metabolites. The goal of this work was to isolate the natural products responsible for induction activity using bioassay-guided fractionation of the organic extract of the brown seaweed Stypopodium zonale and the attachment of juveniles of the common brown mussel, Perna perna, as a model. The meroditerpene epitaondiol, identified by comparison of spectral data with the literature, promoted as much as 4.7 times more mussel attachment compared to controls at the natural concentration found in this alga (0.041% of the crude extract or 0.012% of algal dry weight). This is the first report showing that a seaweed produces terpenoid compounds as cues for invertebrate attachment, and future studies evaluating this action on settlement of mussels in the field are expected to improve aquaculture technology by increasing mussel spat production.     

Isolation and Purification of Cytokinin-Binding Protein from Wheat Chloroplasts

The extract of wheat chloroplast membrane proteins was precipitated by different saturation of (NH4)2SO4. Pellet of 0–30% saturation showing high binding activity to 3H-6BA wax loaded on the affinity chromatography column which was prepared by coupling 6BA to epoxy activated sepharose 6B. The CTK-binding protein was eluted from the BA-sepharose 6B column with Tris buffer containing 0.1 mmol/L 6BA. It showed a single protein band on PAGE and the apparent molecular weight was about 250kD. Two bands with molecular weight of 60kD and 66kD were detected on SDS-PAGE. It was supposed that the protomer of CTK-binding protein was a tetramer of two subunits.     

从基因工程菌分离纯化心钠素

以高效表达的基因工程菌为材料,对以包涵体形式存在的目的产物——心钠素（ＡＮＦ）融合蛋白进行了酶切及分离纯化．先用ＴｒｉｔｏｎＸ－１００和低浓度尿素、盐酸胍洗涤,提高包涵体的纯度．再用高浓度的尿素处理,使蛋白变性溶解,脱氧胆酸钠对蛋白有明显的促溶作用．解决变性蛋白质去除变性剂时的聚合问题．在非变性条件下进行ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＦＰＬＣ系统纯化,得到目的融合蛋白．用凝血酶对融合蛋白作特异性切割,释放出ＡＮＦ小肽,经ＦＰＬＣ分离鉴定,与标准ＡＮＦ有相同的ＴＲ值,说明其纯度已达均一．生物活性检测表明：有明显的放免活性和舒张血管、利尿的生理活性     

海藻多糖抑制白细胞呼吸爆发作用研究

采用ESR、自旋捕集及自旋氧探针技术,研究了海藻硫酸多糖(SPS)对豆蔻酰佛波醇乙酯(PMA)刺激的多形核白细胞(PMN)呼吸爆发的影响.结果表明,SPS能显著抑制PMN呼吸爆发,10 g/L和5 g/L SPS几乎完全清除PMN呼吸爆发产生的自由基,1 g/L SPS可清除53.2%;10 g/L SPS对PMN的耗氧量也有较明显的抑制作用.     

日粮添加海藻粉和抗菌肽对海兰褐蛋鸡产蛋性能的影响

采用4×2（海藻粉×抗菌肽）完全随机试验设计,研究日粮添加不同水平的海藻粉（0．0％、1．0％、3．0％、5．0％）和抗菌肽（300 mg/kg、600 mg/kg）对蛋鸡不同产蛋期（24～27周龄和28～31周龄）产蛋性能的影响。结果表明：（1）日粮海藻粉的添加水平显著提高了产蛋率和料蛋比（P＜0．05）。（2）抗菌肽添加水平在28～31周龄显著提高了产蛋率（P＜0．05）。（3）海藻粉和抗菌肽互作效应显著提高了产蛋率和料蛋比（P＜0．05）。结果提示,海藻粉和抗菌肽对蛋鸡产蛋性能的影响存在互作效应,以海藻粉5％、抗菌肽300 mg/kg水平组合最为理想。     

孔石莼(Ulya pertusa)凝集素的分离纯化及性质的研究

为抑制肿瘤细胞增殖和防治有关病害提供基础理论依据,将孔石莼(Ulva pertusa)经磷酸盐缓冲液抽提,20%～75%硫酸铵分级沉淀,牛甲状腺球蛋白-Sepharose 4B亲和层析,可以从绿藻孔石莼中纯化出孔石莼凝集素(UPL),在PAGE上显示单一蛋白染色带,在等电聚焦电泳上显示单一蛋白染色带,其pI为8.40.纯化后的UPL的最大紫外吸收峰在285 nm,用Sephadex G-200分子筛层析测得其分子量为11 047.该凝集素可以凝集人的A、B、AB、O型红细胞,且凝集活性相同,在对人(A、B、AB、O)兔、鲤、鲫的红细胞的凝集作用中,兔的凝集作用最强.该凝集素凝集兔红细胞的作用不被D-半乳糖、D-果糖、葡萄糖、蔗糖、甘露聚糖、γ球蛋白、卵清蛋白所抑制,仅被牛甲状腺球蛋白抑制,最小抑制浓度为6.20 g/L.该凝集素在pH4.0～10.14范围内均有活性,但在pH6.50～9.51范围内活性较高,该凝集活性在85℃加热1 h,活力仍未改变,说明具有很强的耐热性.