Biliprotein assembly in the hemidiscoidal phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus Cohn. Characterization of dissociation products with special reference to the peripheral phycoerythrocyanin-phycocyanin complexes

The dissociation products of isolated phycobilisomes of Mastigocladus laminosus were separated and analyzed by ultracentrifugation and, in part, by isoelectric focusing. With the exception of the allophycocyanin core, the sedimentation constants of peripheral phycocyanin- and phycoerythrocyanin-phycocyanin complexes lay in the range of 6 to 17S. The latter was represented by a 17S aggregate of two hexameric phycocyanins (dodecamer, dipartite unit). A complex with an absorption maximum at 610 nm (phycocyanin) and a shoulder at 580 nm (phycoerythrocyanin), a fluorescence emission maximum at 645 nm and a sedimentation constant of 11 S is described as a heterogeneously composed hexamer of ()₃-phycoerythrocyanin-()₃-phycocyanin. It was stable under extended dissociation in the cold and under isoelectric focusing. An aggregate of 14 S with an absorption maximum at 576 nm and a shoulder in the fluorescence emission spectrum at 625 nm (phycoerythrocyanin) in addition to the maximum at 645 nm (phycocyanin) is interpreted as a polar phycoerythrocyanin/ phycoerythrocyanin-phycocyanin complex. Combining these complexes with phycocyanin dodecamers creates peripheral rods of the phycobilisome. A proposal of the phycobiliprotein distribution within the phycobilisome of M. laminosus is presented.Abbreviations APC allophycocyanin - PC phycocyanin - PE phycoerythrin - PEC phycoerythrocyanin     

Characterization and structural properties of the major biliproteins of Anabaena sp.

Studies are presented of the biliproteins of Anabaena sp. This filamentous cyanobacterium contains three major biliproteins. Whereas two of these, C-phycocyanin and allophycocyanin, are common to all cyanobacteria, the third, phycoerythrocyanin (_max568nm) has hitherto not been described and its distribution among cyanobacteria appears to be limited. Anabaena variabilis and Anabaena sp. 6411 allophycocyanin, C-phycocyanin, and phycoerythrocyanin were purified to homogeneity and characterized with respect to molecular weight, isoelectric point, absorption spectrum and amino acid composition. The and subunits of each of these proteins were also purified to homogeneity and characterized in the same manner. The tetrapyrrole chromophore content was determined for each of the proteins and subunits. The subunit of phycoerythrocyanin carries a novel phycobiliviolin-like chromophore. This chromophore has not previously been detected in cyanobacterial biliproteins, but has been noted as a prosthetic group of a cryptophytan phycocyanin.Sedimentation equilibrium studies show that at pH 7.0, at protein concentrations of 0.2–0.6 mg/ml, allophycocyanin, C-phycocyanin and phycoerythrocyanin, each exists as a trimeric aggregate, ()₃, of molecular weight of approximately 105000. Structural studies of microcrystals of these three biliproteins by electron microscopy and X-ray diffraction reveal a common plan for the construction of higher assembly forms. The major building block appears to be the trimer ()₃. It is proposed that this is a dise-like structure about 3.0×12.0 nm. The individual or subunits are roughly spherical, 3 nm in diameter. Allophycocyanin trimers stack to form bundles of rods which form long needles. Both phycocyanin and phycoerythrocyanin form double dises ()₆ which are visible as ring-shaped structures by electron microscopy. The mode of assembly of the biliproteinstructures in the phycobilisome is, as yet, unknown.Abbreviation Used SDS sodium dodecyl sulfate Dedicated to Prof. Dr. Roger Y. Stanier on the occasion of his 60th birthday.     

优化培养基增加层理鞭枝藻藻胆蛋白产量

藻胆蛋白(phycobiliprotein)是蓝藻和红藻藻胆体的组成部分,是光合作用集光复合体的组成部分,一般由α和β亚基构成,每个亚基含1～4个辅基色素,从而使藻胆蛋白具有特定的光谱吸收性质。根据这些吸收光谱性质,可以将藻胆蛋白分为:别藻蓝蛋白(APC)、藻蓝蛋白(PC)和藻红蛋白(PE)等,在某些缺乏PE而有异形胞的蓝藻中存在充当PE天线捕光功能的藻红蓝蛋白(PEC)〔1〕。藻胆蛋白可用于天然食用色素、化妆品色素和制药行业,还可作为免疫检测、荧光显微技术和流式细胞荧光测定法技术方面的荧光探针。特别是本工作研究的层理鞭枝藻(简称M.laminosu…     

Isolation and biliprotein characterization of phycobilisomes from the thermophilic cyanobacterium Mastigocladus laminosus Cohn

A method for the effective isolation of functionally intact phycobilisomes from the thermophilic cyanobacterium M. laminosus is presented, using an unconventional high buffer molarity for stabilizing the aggregates and introducing a DNAse treatment of the disrupted cells to obtain sharp banding of the phycobilisomes in the linear sucrose density gradients.The structural integrity of the isolated phycobilisomes is demonstrated by a fluorescence emission maximum at 673 nm of aggregated allophycocyanin and by electron microscopy.Besides C-phycocyanin and allophycocyanin, phycoerythrocyanin is a constituent pigment of the phycobilisomes. These pigments indicated in the absorption spectrum of phycobilisomes with a maximum at 610 nm and two shoulders at 650 and 580 nm, respectively, were characterized by spectral data and isoelectric points.     

Intermediate binding of phycocyanobilin to the lyase, CpeS1, and transfer to apoprotein

The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300–14305). We now show that CpeS1 can bind PCB, as assayed by Ni²⁺ chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower (∼1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1–PCB complex.     

Purification and initial characterization of phycobiliproteins from the endophytic cyanobacterium of Azolla

The endophytic cyanobacterium, Anabaena azollae, isolated from laboratory cultures of Azolla caroliniana Willd., contains three spectroscopically distinct biliproteins. About 70% of the biliprotein is c-phycocyanin (_max 610 nm) and 13% is allophycocyanin (_max 647 nm, shoulder 620 nm). A third pigment corresponds to phycoerythrocyanin (_max 570 nm, shoulder 590 nm). In very dilute solutions of allophycocyanin, at constant pH and buffer strength, the 647 nm maximum disappears and a single _max occurs at 615–620 nm. The 647 nm absorption maximum reappears upon concentrating the dilute solution. Very dilute solutions of phycoerythrocyanin exhibit a broad peak between 570 and 590 nm. Absorption spectra of c-phycocyanin are not significantly altered upon dilution. Fluorescence emission maxima of phycoerythrocyanin, c-phycocyanin, and allophycocyanin occur at 630 nm, 643 nm and 660 nm respectively, using 540 nm excitation. Two subunits, of molecular weight 16,500 () and 20,600 (), are seen in c-phycocyanin upon dissociation with SDS. Dissociation of allophycocyanin and phycoerythrocyanin with SDS yields one sizeclass of subunits, with a molecular weight of about 17,500 for allophycocyanin and 18,000 for phycoerythrocyanin.Contribution No. 684 Offprint requests to: G. A. Peters     

New horizons in culture and valorization of red microalgae

Research on marine microalgae has been abundantly published and patented these last years leading to the production and/or the characterization of some biomolecules such as pigments, proteins, enzymes, biofuels, polyunsaturated fatty acids, enzymes and hydrocolloids. This literature focusing on metabolic pathways, structural characterization of biomolecules, taxonomy, optimization of culture conditions, biorefinery and downstream process is often optimistic considering the valorization of these biocompounds. However, the accumulation of knowledge associated with the development of processes and technologies for biomass production and its treatment has sometimes led to success in the commercial arena. In the history of the microalgae market, red marine microalgae are well positioned particularly for applications in the field of high value pigment and hydrocolloid productions. This review aims to establish the state of the art of the diversity of red marine microalgae, the advances in characterization of their metabolites and the developments of bioprocesses to produce this biomass.     

鱼腥藻PCC7120藻红蓝蛋白α亚基体内重组研究

为了研究藻红蓝蛋白α亚基的生物合成途径,通过构建相容的4种重组质粒pETDuetp-ecA、pCOLADuet-pecE、pCDFDuetp-ecF和pACYCDuet-ho1-pcyA,将裂合酶基因pecE和pecF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻红蓝蛋白α亚基基因pecA共同转入大肠杆菌BL21(DE3),通过色素蛋白锌电泳和光谱检测表明产生了生物活性的PecA-PCB。结果表明生成的色素藻胆蛋白具有藻红蓝蛋白α-亚基所特有的光谱性质和可逆光致变色性质。而在裂合酶基因pecE和pecF不转入大肠杆菌的情况下,大肠杆菌内只有0.1%的PecA-PCB产生。以上研究对藻胆蛋白生物构建具有重要意义。