碳和氮代谢被抑制诱导雨生红球藻细胞内虾青素的合成

为研究雨生红球藻(Haematococcus pluvialis)合成虾青素的机理,文中分析了不同诱导条件下藻细胞内氮和碳代谢的变化。结果表明:强光照(HL)、添加乙酸钠(AA)、缺氮(NF)和缺磷(PF)都直接或间接地影响了雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶(Rub isco)和硝酸还原酶(NR)的活性,导致2种酶的活性大幅度下降。只有当Rub isco和NR的活性降到非常低的水平时,藻细胞才开始合成虾青素。与此相反,对照(CK)中这2种酶的活性一直较高,但细胞内没有虾青素积累。由于Rub isco和NR是雨生红球藻碳代谢和氮代谢的关键酶,因此碳和氮代谢被抑制是诱导雨生红球藻合成虾青素的原因。     

利用雨生红球藻生产虾青素的小试研究

以BG11为基本培养基,小试研究了环境因子的调控对雨生红球藻(Haematoccus pluvialis,FACHB-712)生长和虾青素积累的影响。通过单因子实验改变营养方式、pH值、温度、光照时间和光照强度,测定藻液光密度和虾青素含量等指标。结果表明:添加1g/L葡萄糖作为碳源可明显提高藻种生物量;pH=8.0左右,22～27℃,高光强(200μmol·m-2·s-1)连续光照(24h)的生长条件最适合雨生红球藻游动细胞的增殖,可以作为大规模培养雨生红球藻生产虾青素的实验依据。     

强光照对雨生红球藻合成虾青素的影响

在不同的光照强度下研究了雨生红球藻细胞内虾青素的合成与初级代谢的关系.在强光(HL)和中等强度(ML)的光照条件下,雨生红球藻细胞内1,5-二磷酸核酮糖羧化酶 (Rubisco)和硝酸还原酶(NR)的活性第1天大幅度提高,2天后又迅速下降.与此同时,硝酸盐浓度也快速降低.当虾青素在第4天(HL)和第6天(ML)开始合成时, HL中Rubisco和NR活性以及NO3-浓度分别下降了75.5%,71.5% 和96.2%,而ML中则下降了76.5%,74.7% 和94.3%.相比之下,在低光照(LL)条件下,实验结束时三个指标仅下降了25.9%,29.8% 和56.8%,细胞中没有虾青素积累.结果表明强光提高了Rubisco 和 NR活性,导致硝酸盐浓度迅速降低而最终又抑制了这两种酶的活性,造成雨生红球藻光合作用效率下降即"碳饥饿".在此状态下,为了生存,细胞内合成虾青素的相关基因被激活,藻细胞开始合成并积累虾青素.     

从雨生红球藻藻泥中选择性提取虾青素

以雨生红球藻湿藻泥为原料,研究了不同有机溶剂对胞内油脂和虾青素选择性提取分离的影响,通过酸解破壁提高虾青素和油脂的提取效率。结果表明,连续乙醇提取可对胞内色素和油脂有效分级提取,先提取出极性组分(叶绿素和极性脂),再提取中性组分(类胡萝卜素和中性脂)。中等极性溶剂或溶剂体系对类胡萝卜素的选择性和提取率较好;乙醇/乙酸乙酯混合溶剂提取类胡萝卜素的总得率(干重)达25.31 mg/g,提取率为69.35%。对雨生红球藻湿细胞进行酸解破壁处理有助于提高虾青素和油脂的提取率。在最优酸解破壁条件(盐酸浓度1 mol/L,温度60℃,时间60 min)下,含水80%的雨生红球藻藻泥的油脂总得率(干重)达418 mg/g,总脂提取率达97%。     

转光膜在雨生红球藻养殖上的应用

通过对雨生红球藻在不同光质条件下生长的比较,确定了红色光有利于藻生长,进而用2.5 L气升式光照反应器在转光膜及普通PE膜下培养藻进行对比,结果显示雨生红球藻生物量、色素、光合活性等几项生物指标在转光膜条件下明显高于普通PE膜. 在气升式反应器内培养的藻细胞,接种9 d,虾青素含量可达3.57 mg/L,叶绿素浓度达到12.42 mg/L,干重提高8.8%以上.     

两种培养基间雨生红球藻细胞生长分化差异及磷的作用

基于雨生红球藻游动细胞和不动细胞间Triton X-100敏感性的差异,建立了一种快速监测培养过程中细胞分化的指标,对比了雨生红球藻在BG11和BBM两种培养基中的细胞生长与分化差异. 结果表明,在BBM培养基中培养8 d,雨生红球藻的游动细胞比例始终较高,而在BG11中早期不动孢子囊释放的游动孢子则快速成熟并转变成不动细胞. 这主要是由于BBM中游动细胞通过有丝分裂或孢子囊两种模式维持了其继续分裂与增殖的能力,而BG11中这些繁殖形式缺失的主要原因在于其较低的磷浓度,培养基中适当的高磷含量对保持雨生红球藻培养中期游动细胞的持续繁殖非常关键.     

从雨声红球藻中提取虾青素的皂化工艺优化

目的:优选从雨声红球藻提取虾青素的皂化工艺。方法:以虾青素的提取得率为考察目标,通过单因素法、正交设计法优化从雨声红球藻提取虾青素的皂化工艺。结果:筛选最佳皂化工艺为是加入10倍雨生红球藻量的4%KOH无水乙醇溶液,在10℃下皂化10min。结论:筛选出来的皂化工艺稳定可行、操作简单。     

雨生红球藻合成虾青素过程中分泌铵离子的研究

雨生红球藻在在缺氮(NF)的条件下,随着虾青素的合成,肽链内切酶(EP)的活性上升而蛋白质含量下降,天冬酰氨酸含量在前4天增加然后又减少,铵离子浓度则持续上升。相比之下,在氮充足(NR)的培养液中,藻细胞不合成虾青素,蛋白质和天冬酰氨酸的含量以及EP的活性基本稳定,培养液中也没有检测到铵离子。上述结果表明:①降解的蛋白质为虾青素的合成提供了碳源,②EP参与了蛋白质的降解反应,③为避免铵离子的毒害作用,蛋白质降解所产生的部分氨临时贮存在天冬酰氨酸中,而其余的则分泌到细胞外。     

盐胁迫诱导雨生红球藻合成虾青素的机理

为研究盐胁迫诱导雨生红球藻合成虾青素的机理,分析了在添加氯化钠(HS)和未添加氯化钠(CK)的培养液中,细胞内氮和碳代谢的变化。结果表明:HS中硝酸还原酶(NR)和1,5-二磷酸核酮糖羧化酶(Rub isco)活性迅速下降。虾青素在第4天开始合成时,二者分别降至初始值或最高值的46.5%和25.7%。相比之下,在对照(CK)中,NR和Rub isco活性仍然很高,仅下降了26.1%和25.6%,细胞内没有虾青素积累。上述数据表明盐胁迫条件下NR活性被抑制,细胞内氮源供应不足(氮饥饿)并进一步抑制了Rub isco的合成,导致CO2固定量减少(碳饥饿)。为了生存,藻细胞开始合成虾青素。     

虾青素的生产方法

近年来,虾青素作为高级营养保健品和药品的潜力倍受关注,其产品定位主要在强化免疫系统功能、抗癌、抗炎、保护视网膜免受紫外线辐射和光氧化损伤等方面。 虾青素的生产方法一般有如下几种：从水产品加工废弃物中提取虾青素、利用藻类如雨生红球藻等生产虾青素、利用红法夫酵母发酵生产虾青素和化学合成法等等。     

Isolation and characterization of thermostable phycocyanin from Galdieria sulphuraria

Phycocyanin is a highly valuable pigmented protein synthesized by several species of cyanobacteria and red alga. In this study we demonstrate the production of thermostable phycocyanin from the unicellular red alga Galdieria sulphuraria. Phycocyanin was extracted by repeated freeze-thaw cycles and purified in a two-step process using ammonium sulfate fractionation, at 25% and 50% concentrations. Purified phycocyanin exhibited maximum absorbance at 620 nm, and the purity ratio (A₆₂₀/A₂₈₀) was found to be greater than 4. The recovery efficiency of phycocyanin from the crude extract was above 80%. In total, approximately 19 milligram pure phycocyanin was obtained from 3 g of wet cell mass of Galdieria sp. Subunits α and β of the protein were separated by SDS-PAGE and analyzed by MALDITOF mass spectrometry for identification, which confirmed that the isolated protein is phycocyanin. The molecular weight of α and β subunits of phycocyanin was found to be 17.6 and 18.4 kDa, respectively.     

The release of the blue biological pigment C‐phycocyanin through calcium‐aided cytolysis of live Spirulina sp.

C‐phycocyanin is a light‐harvesting phycobiliprotein found in, amongst other species, the cyanobacterium Spirulina sp. C‐phycocyanin is bright blue in colour and can be used as a natural blue colorant for a variety of applications. Various cell disruption methods exist to cause the lysis of the cyanobacterial cells and release of phycocyanin, but these methods have significant drawbacks, such as cost, difficulty in scale‐up, bacterial contamination, or risk of degrading the protein. This article outlines an alternative method for cell disruption based on the use of Ca(II), which lyses live Spirulina biomass, releasing phycocyanin at a range of concentrations (0.1–0.8 m ) over varying time periods, depending on the conditions. In comparison with other ionic species tested, Ca(II) performed best by a significant margin. Bead milling of biomass was used to quantify the maximum phycocyanin in the biomass, and the greatest was found to be ca. 90% released into solution after 48 h under 0.5 m Ca(II) in a 0.35 m acetate buffer at pH 6. Exposing Spirulina to sodium azide revealed that the mechanism of Ca(II) ion‐aided cytolysis is likely based on a metabolically driven process. This study demonstrates a potential processing option for the release of phycocyanin from live Spirulina, paving the way for the development of a novel bioprocess for the industrial production of the biological pigment.     

Practical application of aqueous two‐phase systems for the development of a prototype process for c‐phycocyanin recovery from Spirulina maxima

A novel process for the recovery of c‐phycocyanin from Spirulina maxima exploiting aqueous two‐phase systems (ATPS), ultrafiltration and precipitation was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the c‐phycocyanin and contaminants concentrate to opposite phases. PEG1450–phosphate ATPS proved to be suitable for the recovery of c‐phycocyanin because the target protein concentrated in the top phase whilst the cell debris concentrated in the bottom phase. A two‐stage ATPS process with a phase volume ratio (V_r) equal to 0.3, PEG1450 7% (w/w), phosphate 20% (w/w) and system pH of 6.5 allowed c‐phycocyanin recovery with a purity of 2.4 (estimated as the relationship of the 620 nm to 280 nm absorbances). The use of ultrafiltration (with a 30 kDa membrane cut‐off) and precipitation (with ammonium sulfate) resulted in a recovery process that produced a protein purity of 3.8 ± 0.1 and an overall product yield of 29.5% (w/w). The results reported here demonstrated the practical implementation of ATPS for the design of a prototype recovery process as a first step for the commercial purification of c‐phycocyanin produced by Spirulina maxima. © 2001 Society of Chemical Industry     

含吡啶环双元噁二唑衍生物的合成及其光谱性质和电化学性质

合成了6个含吡啶环的双元噁二唑化合物,产率51%~78%。用1HNMR、FTIR、MS对其结构进行了表征。考察了它们的UV-Vis吸收光谱、荧光光谱,并用循环伏安法测定了其电化学性能。吸收光谱表明:a~c能有效地形成共轭,λmax分别为278、309、282 nm,d~f的共轭效应差,吸收光谱基本相同,λmax分别为273、272 nm和270 nm。荧光光谱表明:目标化合物的DMF溶液发射强的紫色荧光,a和b最大发射波长为398 nm和396 nm,c蓝移到364 nm;d~f出现双发射峰,d和e最大发射峰都在334 nm和345 nm,f红移到342 nm和356 nm,而固体发射强的紫蓝色荧光,在376~414 nm出现最大发射峰;电子亲和势为2.57~3.11 eV,离子势为5.97~6.70eV,说明目标化合物的电子传输性能良好(PBD:EA=2.82 eV)。     

New Generation of Low Pressure Mercury Lamps for Producing Ozone

Producing ozone by means of a low pressure mercury discharge is still limited by such economical and technical factors as efficiency and lifetime of Low Pressure Mercury Lamps (LPMLs). With the introduction of a new “long life coating” technology for LPMLs at Heraeus, these lamps show up as an effective and reliable VUV-light source that can be used in ozone generation. Different coating technologies are compared in terms of radiation losses in the coating and depreciation of the mercury resonance line at λ=185 nm. The parameters of new ozone generating lamps are presented. A simple model with consideration of both the resonance lines at λ=185 nm and λ=254 nm for a practical calculation of ozone concentration in an air flow is proposed.