Extracellular Carbohydrates and Polysaccharides of the Alga Chlorella pyrenoidosa Chick S-39

In the growing culture of the thermophilic alga Chlorella pyrenoidosa Chick S-39, the amount of extracellular carbohydrates in the medium reached 5–17% of their content in the cells and 20–40% of the total content of extracellular organic matter. Experiments with the enrichment and synchronous algal cultures showed that the accumulation of extracellular carbohydrates and polysaccharides in the media occurred due to their release from the cells, rather than to cell lysis, and depended on cell photosynthetic activity and reproduction. Chromatographic determination of free sugars revealed the presence of saccharose, glucose, and fructose in the culture medium. Extracellular carbohydrates in C. pyrenoidosa cultures were represented mainly by water-soluble polysaccharides containing galactose, mannose, arabinose, xylose, ribose, fucose, and rhamnose.     

Characterization of Two Extracellular Polysaccharides from Marine Bacteria

Two bacterial isolates from the intertidal zone produced significant quantities of extracellular polysaccharide with interesting properties. One polysaccharide was named PS 3a24; the other was named PS 3a35. The relative proportion of sugars in PS 3a35 was 51.6% glucose, 39.0% galactose, 3.1% mannose, and 6.3% rhamnose, with a trace of an unidentified sugar. PS 3a24 was composed of 40.2% glucose, 57.2% galactose, and 2.6% mannose. PS 3a35 contained 6% pyruvate, whereas PS 3a24 contained no pyruvate. Both exhibited high specific viscosity, pseudoplasticity, and stability over a wide range of pH in the presence of a variety of salts. The viscosity of PS 3a35 was relatively insensitive to increasing temperature, whereas that of PS 3a24 showed an irreversible drop on heating.     

发状念珠藻胞外多糖的纯化与性质分析

采用DEAE阴离子交换层析和Sephadex G100凝胶层析对液体悬浮培养发状念珠藻胞外多糖进行纯化, 得到两个组分NFPS1和NFPS2。对组分NFPS2进行理化性质分析, 并与野生发状念珠藻多糖NFPS0的性质进行对比。结果表明二者具有相似的单糖组成, 均为葡萄糖、木糖、半乳糖、甘露糖; 表观分子量分别为2.79×105、2.26×105; 均不含核酸、蛋白质等物质, 是非硫酸化多糖; 有较高的热稳定性, 其降解温度在245oC左右。但在微观结构上, 两者存在一定差别。     

发状念珠藻胞外多糖的纯化与性质分析

采用DEAE阴离子交换层析和Sephadex G100凝胶层析对液体悬浮培养发状念珠藻胞外多糖进行纯化, 得到两个组分NFPS1和NFPS2。对组分NFPS2进行理化性质分析, 并与野生发状念珠藻多糖NFPS0的性质进行对比。结果表明二者具有相似的单糖组成, 均为葡萄糖、木糖、半乳糖、甘露糖; 表观分子量分别为2.79×105、2.26×105; 均不含核酸、蛋白质等物质, 是非硫酸化多糖; 有较高的热稳定性, 其降解温度在245oC左右。但在微观结构上, 两者存在一定差别。     

An Arabinogalactan from Extracellular Polysaccharides of Suspension-cultured Tobacco Cells

The structure of an arabinogalactan, separated from extracellular polysaccharides of cultured tobacco cells, has been investigated by methylation analysis of the original polysaccharide and of the products obtained after mild acid hydrolysis and after controlled Smith degradation.

The arabinogalactan consists of l-arabinose, d-galactose and l-rhamnose in the molar ratio of 47: 45: 8. The arabinogalactan has a main chain of (1→3)-linked d-galactopyranosyl residues, half of which are substituted at the 6-position. Most of the side chains consist of three (1→6)-linked D-galactopyranosyl residues, to which l-arabinose residues are attached at C-3. The l-arabinofuranosyl and pyranosyl residues are present as end groups, and l-arabinopyranosyl residues are attached to C-5 of l-arabinofuranosyl residues. Non-reducing terminal l-rhamnopyranosyl residues are also present.     

Biodegradability of Food-Associated Extracellular Polysaccharides

Exopolysaccharides (EPSs) produced by lactic acid bacteria, which are common in fermented foods, are claimed to have various beneficial physiological effects on humans. Although the biodegradability of EPSs is important in relation to the bioactive properties, knowledge on this topic is limited. Therefore, the biodegradability of eight EPSs, six of which were produced by lactic acid bacteria, was compared with microorganisms from human feces or soil. EPS-degradation was determined from the decrease in polysaccharide-sugar concentration and by high-performance size exclusion chromatography (HPSEC). Xanthan, clavan, and the EPSs produced by Streptococcus thermophilus SFi 39 and SFi 12 were readily degraded, in contrast to the EPSs produced by Lactococcus lactis ssp. cremoris B40, Lactobacillus sakei 0-1, S. thermophilus SFi20, and Lactobacillus helveticus Lh59. Clearly, the susceptibility of exopolysaccharides to biological breakdown can differ greatly, implying that the physiological effects of these compounds may also vary a lot. Received: 23 August 1999 / Accepted: 5 October 1999     

小球藻的优化培养及胞内多糖的提取

小球藻细胞中所含有的多糖和糖蛋白等活性物质具有明显的抗肿瘤,抗病毒感染及增强机体免疫力等作用。首先考察了几个重要的环境因素对小球藻生长及胞内多糖含量的影响。确定了优化的环境条件为：光照17h,培养温度15℃,营养盐KNO3浓度为2mmol/L。在此优化条件下,大批量培养小球藻至平衡期,离心收集藻细胞,再经过冻融与超声波破碎,三氯乙酸沉淀蛋白质,最后通过SephadexG-75凝胶柱分离得到了两种分子量不同的多糖,并结合醋酸纤维素膜电泳证明了同样的结论。     

Extracellular proteases of Staphylococcus spp

Bacterial proteases secreted into an infected host may exhibit a wide range of pathogenic potentials. Staphylococci, in particular Staphylococcus aureus, are known to produce several extracellular proteases, including serine-, cysteine- and metalloenzymes. Their insensitivity to most human plasma protease inhibitors and, even more, the ability to inactivate some of these make the proteases potentially harmful. Indeed, several recent studies have shown that staphylococcal proteases are able to interact with the host defense mechanisms and tissue components as well as to modify other pathogen-derived virulence factors. A tight, cell density-dependent control of proteolytic activity expression, similar to that of the well-defined virulence determinants, further suggests the role of staphylococcal proteases in the infection process. Consistently, alterations in coordinated expression of extracellular proteins markedly diminished the virulence. However, despite these data and the fact that a strain deficient in sspABC operon coding for serine (sspA) and cysteine (sspB) proteases was highly attenuated in virulence in the animal infection model, it was impossible to unambiguously demonstrate the importance of any particular protease as a virulence factor. Therefore, it can be assumed that the orchestrated expression and interaction of a variety of extracellular and cell surface proteins rather than any particular one is responsible for the staphylococcal pathogenicity and that the proteases apparently play an important role in this complex process. Such redundant mechanism is very well suited for promoting the survival of staphylococci under diverse environmental conditions encountered in the infected host.     

3种小球藻DNA提取方法的比较

单细胞真核藻类小球藻已成为藻类分子生物学研究的热点,但其特殊的细胞壁组分给DNA的提取带来了一定的困难。文章比较了3种从小球藻中提取DNA的方法,即纤维素酶法、作者改进的CATB法和目前单细胞藻类常用的裂解法。通过所提取的DNA进行浓度、纯度分析,认为CTAB法是一种简单、快速和高效的适合于小球藻的DNA提取方法。     

小球藻生物富集锌、镉机制的研究

藻类对许多重金属具有较强的生物富集能力,小球藻（Chlorella spp.)分别在不同系列Zn^2 ,Cd^2 浓度下培养8d间隔2d,测定小球藻的生物量及Zn和Cd的含量。结果表明,小球藻随水体中Zn^2 和Cd^2 的浓度不同,在重重金属水体中暴露时间不同,具有不同的富集速率和能力。     

The Kinetics of Extracellular Glycollate Production by Chlorella pyrenoidosa

Extracellular glycollate is liberated by Chlorella pyrenoidosaduring growth in medium bubbled with air or 3 per cent carbondioxide in air. With air the rate of release of glycollate percell decreases, with 3 per cent carbon dioxide it increases,with increase in cell number. Glycollate is released duringshort-term experiments when C. pyrenoidasa, grown under lowlight and high carbon dioxide, is transferred suddenly to highlight and low carbon dioxide. No other combination of thesefactors produces a comparable release of glycollate. The quantityof glycollate released in short-term experiments increases exponentiallywith the relative growth-rate of the culture from which thecells are derived. A crucial condition for maximum glycollaterelease is that growth of the culture prior to the experimentshould not be limited by carbon-dixoide concentration. The effectof pH is related to its effect on growth-rate; i.e. C. pyrenoidosahas a lower relative growth-rate at pH 8.3 and produces correspondinglyless glycollate than faster growing cultures at pH 6.4. Duringshort-term experiments under high light and low carbon dioxidethe rate of glycollate release drops after 50–100 minutessuggesting exhaustion of the glycollate precursor.     

溶藻细菌胞外活性物质对蛋白核小球藻的毒性效应

为了探索分离到的溶藻细菌L7胞外活性物质对蛋白核小球藻的毒性效应和致毒机理, 采用不同质量浓度的L7胞外活性物质冻干粉(L7-LPEAC)处理蛋白核小球藻(Chlorella pyrenoidosa), 测定藻细胞的光合作用效率(EPR)以及蛋白质、叶绿素a和丙二醛(MDA)的含量。L7-LPEAC溶液浓度较低(0.80 g/L和1.25 g/L)时促进蛋白核小球藻的生长, 其96 h、120 h的EC50分别为5.75 g/L和2.55 g/L。当L7-LPEAC溶液浓度≥2.00 g/L时, 叶绿素a和蛋白质含量变化同步呈现先增加后减少的趋势; 处理72 h后, L7-LPEAC溶液浓度分别为2.00 g/L, 3.13 g/L, 4.90 g/L的浓度组中, 藻细胞MDA含量与对照组相比差异显著(P<0.05), 浓度组7.67 g/L和12.00 g/L则与对照组差异极显著(P<0.01); 处理120 h后, 各浓度组藻细胞叶绿素a含量的相对抑制率均大于60%。使用L7-LPEAC修复富营养化水体时, 选择适当的投加浓度, 既能杀灭引起水体富营养化的目标藻类, 又能避免对其他藻类产生抑制作用, 可以较好地维持水生生态系统的平衡。     

海南红树林淡紫拟青霉胞外多糖提取条件的优化

为了探讨影响红树林淡紫拟青霉胞外多糖提取的因素,确定最佳提取方案,设置不同的发酵液浓缩倍数、三氯乙酸用量等因素,设计单因素实验测定多糖提取最佳条件。然后设计正交试验,检测多糖在不同条件下的提取率,以获得最佳提取工艺。结果发现,发酵液浓缩3~5倍时多糖提取率最高,10%的三氯乙酸对蛋白质脱除效果最好;正交试验表明影响多糖提取的因素依次为乙醇用量、沉淀时间和温度,最优方案为3倍95%乙醇、4 ℃沉淀24 h。该条件下,多糖提取率可达57.835%±1.206%。研究结果为红树林淡紫拟青霉胞外多糖的提取研究提供了参考。     

铜绿假单胞菌Psl多糖转运蛋白PslD的表达与纯化研究

条件性致病菌铜绿假单胞菌是细菌生物被膜研究的模式菌,其分泌的胞外多糖Psl在生物被膜形成中起关键作用。PslD为Psl多糖的转运蛋白,由256个氨基酸构成,生物信息学分析揭示其有N端信号肽且为一次跨膜蛋白。分离纯化完整跨膜蛋白需要去垢剂的作用,去垢剂种类繁多且性质不一,研究设计了一套筛选溶解PslD的去垢剂的方案。通过抗组氨酸标签的Western blot分析,n-Decyl-β-D-maltopyranoside (DM),n-Decyl-N,N-dimethylamine-N-oxide(DDAO)及n-Dodecyl-N,N-dimethylamine-N-oxide(LDAO)被认为溶解PslD的效率较高。通过改变总蛋白与去垢剂比例,进一步优化了去垢剂的溶解条件即8 mg/mL总蛋白:质量分数为1%的 LDAO。在此溶解条件下,仅通过第一步Ni柱亲和纯化目的蛋白纯度可到达80%以上,这为进一步结晶尝试及其结构生物学研究奠定基础。     

Production and Properties of Extracellular Endoxylanase from Neurospora crassa

Neurospora crassa 870 produced 14 and 0.025 U of extracellular xylanase (1,4-beta-d-xylan xylanohydrolase; EC 3.2.1.8) and beta-xylosidase (1,4-beta-xylan xylohydrolase; EC 3.2.1.37) per ml, respectively, in 4 days when commercial xylan was used as a carbon source. The effects of pH and carbon sources on xylanase production by N. crassa are discussed. Two xylanases (I and II) were purified and had pI values of 4.8 and 4.5 and molecular weights of 33,000 and 30,000. The maximum degree of hydrolysis of xylan by the extracellular culture broth was 66% in 4 h. The end products of xylan hydrolysis by xylanase I and II showed the presence of xylose, xylobiose, xylotriose, xylotetraose, xylopentose, and arabinose, indicating that they are endoxylanases capable of hydrolyzing 1,3-alpha-l-arabinofuranosyl branch points. Both xylanases showed activity toward carboxymethyl cellulose but no activity toward para-nitrophenyl-beta-d-xyloside or laminarin. Xylanase I showed appreciable activity toward para-nitrophenyl-beta-d-glucoside, whereas xylanase II was inactive.     

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)¹ (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself², and is amenable to high-throughput analyses³. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes⁴. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase^{5, 11, 12, 13}, xylan using an endo-β-(1-4)-xylanase ^6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase ⁸. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined ⁹.     

Extracellular enzymatic activity of Malassezia spp. isolates

Extracellular enzymatic activity of different species of Malassezia spp was evaluated. Thirty-three isolates of animal origin (dogs and cats) and stock culture samples were studied. Twenty isolates of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa and one of M. restricta, M. obtusa and M. slooffiae were examined. The enzymatic activity was investigatedusing Api Zym system. The enzymatic patterns showed light differences. Esterase lipase, Phosphatase acid and Naphtol-AS-BI-phosphohydrolase were produced in significant amounts from most isolates excepted for M. restricta, confirming the limited enzymatic activity of this species. Data obtained from the other new species described after the revision of the genus, appear to be quite homogeneous. Dixon’s broth appeared to be a valid medium for the growth of all Malassezia spp. This revised version was published online in June 2006 with corrections to the Cover Date.