Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from selenium-rich algae (Spirulina platensis)

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P2₁ and cell parameters a =108.0 Å , b = 117.0 Å , c = 184.0 Å , β = 90.2° and 12(αβ ) units in the asymmetric unit was obtained by using (NH₄)₂SO₄ as precipitant. These crystals diffract up to 2.8 Å . Form II crystal obtained by using PEG4000 as precipitant belongs to space group P63 with unit cell constants a = 155.0 Å , c = 40.3 Å , γ =120.0° and one(αβ ) unit in the asymmetric unit. The crystals diffract beyond 2.9 Å . The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from selenium-rich algae (Spirulina platen sis)

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chro-matographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P21 and cell parameters a =108.0 A, b = 117.0 A, c = 184.0 A, β = 90.2° and 12(αβ) units in the asymmetric unit was obtained by using (NH4)2SO4 as precipitant. These crystals diffract up to 2.8 A. Form II crystal obtained by using PEG4000 as precipitant belongs to space group P63 with unit cell constants a = 155.0 A, c = 40.3 A, γ=120.0° and one(αβ) unit in the asymmetric unit. The crystals diffract beyond 2.9 A. The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

Thermal degradation kinetics of the phycocyanin from Spirulina platensis

The cyanobacterium Spirulina platensis is a source of pigments, such as phycocyanin, which is used in the food, cosmetic and pharmaceutical industries. The thermal degradation kinetics of the liquid extract at pH values of 5, 6 and 7 was studied, evaluating its stability between 50 and 65 °C. The kinetic model was assumed and validated as being of the first order. Between 50 and 55 °C the extract was more stable at pH 6 and between 57 and 65 °C at pH 5, but was shown to be increasingly unstable at pH 7 as the temperature of the treatment increased. The addition of sorbitol between 10 and 50% (w/w) in the treatment at 62 °C for 30 min increased the half-life values of the phycocyanin extract, proving that its de-colorization was related to degradation of the protein chain.     

Characterisation of a specific Phycocyanin-hydrolysing protease purified from Spirulina platensis

A novel protease has been identified, purified and partially characterised from complete medium grown Spirulina platensis, which could be responsible for the selective proteolysis of phycobiliproteins. It is an 80 kDa homodimeric enzyme; its N-terminal sequence is not related to any known protease sequence. It hydrolyses native phycocyanins in both crude extracts and reconstructed systems with purified Allo- or C-phycocyanin. It is inactive on several native proteins, including ribulose-1,5-bisphosphate carboxylase. The two phycocyanins are degraded at different velocities since C-phycocyanin is the better substrate, in agreement with the earlier observations on the progress of the phycobilisome disassembly. Specificity for synthetic substrates and inhibitors strongly suggests its assignment to the serine-protease family. The enzyme, however, is insensitive to the commercially available protein inhibitors of trypsin-like proteases.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.     

Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Cloning and expression of the genes for ribulose-1,5-bisphosphate carboxylase from Spirulina platensis

The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase have been cloned from the filamentous cyanobacterium Spirulina platensis. The two genes, located very closely on a 4.6 kbp DNA fragment, appear to be expressed although to a different extent in minicells of Escherichia coli. The amount of large subunit produced in the bacterial host represents at least 10% of the total protein.     

Purification,crystallization and preliminary crystallographic studies of a class II chitinase from kidney bean seeds

A new member of class II chitinase from Phaseolus vulgaris was purified and crystallized. Diffraction data to 2.7A resolution have been collected and the preliminary crystallographic studies have been completed. The space group is P1 with unit cell parameters of a=36.32A, b=46.24A, c=70.36A, a =97.9 degrees, b=103.8 degrees and g =110.5 degrees. Molecular replacement and initial refinement statistics indicate there are two chitinase molecules in the crystallographic asymmetric unit.     

Purification,crystallization, and preliminary crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli

The phenylalanine-regulated isozyme of 3-deoxy-D-arabino-heptulosonate-7-phosphate- synthase (DAHPS) from Escherichia coli, its binary complexes with either substrate, phosphoenolpyruvate (PEP), or feedback inhibitor, Phe, and its ternary complexes with either PEP or Phe plus metal cofactor (either Mn²⁺, Cd²⁺, or Pb²⁺) were crystallized from polyethylglycol (PEG) solutions. All crystals of the DAHPS without Phe belong to space group C2, with cell parameters a = 213.5 Å, b = 54.3 Å, c = 149.0 Å, β = 116.6°. All crystals of the enzyme with Phe also belong to space group C2, but with cell parameters a = 297.1 Å, b = 91.4 Å, c = 256.5 Å, and β = 148.2°.     

Purification, crystallization, and preliminary X-ray crystallographic analysis of thermus thermophilus V(1)-ATPase B subunit.

The gene of V(1)-ATPase B subunit from the thermophilic eubacterium Thermus thermophilus has been cloned and the protein overproduced in Escherichia coli. The purified protein, with a molecular weight of 53.2 kDa, was crystallized from 10% (w/v) polyethylene glycol 1000, 120 mM magnesium chloride, and 100 mM Na-tricine, pH 8.0, by the vapor diffusion method. The crystals diffracted X-rays beyond 3.5 A on a synchrotron radiation source. The crystals belong to the monoclinic space group C2, with unit cell dimensions of a = 153.1 A, b = 129.6 A, c = 92.7 A, and beta = 100.3 degrees. Assuming that three or four molecules are contained in an asymmetric unit, the V(M) value is calculated as 2.8 or 2.1 A (3)/Da, respectively.     

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes

Summary A 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elngation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12>EF-Tu>EF-G>S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased.     

钝顶螺旋藻低中温适应型新品系的选育及RAPD分析

【目的】选育低中温适应型螺旋藻新品系,显著拓展螺旋藻工厂化培植的地理范围及时间、提高产量和降低成本。【方法】以用于工厂化培植的钝顶螺旋藻(Spirulina platensis) ZJU0116为出发品系,用组织匀浆和离心分离法制得其单细胞或原生质球,先以0.6%甲基磺酸乙酯(ethyl methanesulfonate, EMS)处理30 min,再用2.4 kGy的⁶⁰Co γ射线辐照,进行低温逆境筛选、5次冷/热(12 ℃/38 ℃)骤变交替处理、藻丝单体分离培养、温度适应面构建和蛋白质含量等检测及生产培植试验。【结果】获得了1株蛋白质含量与ZJU0116的相当、而温度适应面和平均日产量依次提高10.7%和10.9%的低中温适应型突变体,命名为ZJU0116(LMTA)。光学显微形态与随机扩增的多态性DNA (randomly amplified polymorphic DNA, RAPD)分析结果显示,与其亲本ZJU0116相比,ZJU0116(LMTA)藻丝的螺旋数和长度依次减少54.4%和42.1%,螺距增加28.8%;基因组DNA在随机引物S30的扩增产物中多了1条约470 bp的条带。【结论】ZJU0116(LMTA)在工厂化培植中温度适应性好、生产性状稳定,干藻粉产量可增加10%以上,可为当前螺旋藻产业深度融合“双碳”目标、迈向高质量发展新阶段提供必要支撑。     

Purification, crystallization and preliminary analysis of hemoglobin from rabbit (Oryctolagus cuniculus)

Hemoglobin (Hb) is a tetrameric protein, which contains four heme prosthetic groups, and each one is associated with a polypeptide chain. Herein, we report the rabbit hemoglobin which has intrinsically high oxygen affinity and possess highest sequence identity with human hemoglobin. The purified hemoglobin has been tried to crystallize in different crystallization conditions owing to its formation of various crystal systems. The rabbit Hb crystals were grown using PEG 3,350 as the precipitant at 18 degrees C. The crystals of rabbit Hb belongs to triclinic space group P1 with one molecule (alpha2beta2) in the asymmetric unit.     

Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2.

The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A.     

螺旋藻有效成分超临界萃取与分离研究

使用超临界CO2萃取技术对钝顶螺旋藻粉进行萃取与分离,结果表明:超临界CO2萃取的最优条件为压力20Mpa,夹带剂使用量100mL/100g,温度50℃,萃取时间2h;利用凯氏定氮法及气相色谱法分别对超临界萃取处理过的螺旋藻中蛋白质和多糖进行研究与分析,结果表明:螺旋藻粉中蛋白质含量为49.39%,螺旋藻多糖中的单糖含量占多糖粗品的9.25%,主要组成为鼠李糖、木糖、甘露糖、葡萄糖、半乳糖等。     

Probing the mechanism of a cyanobacterial Δ9 fatty acid desaturase from Spirulina platensis C1 (Arthrospira sp. PCC 9438)

The initial and rate determining step in the mechanism of fatty acid desaturases has been proposed to be breakage of one of the C---H bonds at the site of the incipient double bond. This has been investigated and supported for a number of eukaryotic fatty acid desaturases through the use of kinetic isotope effect experiments with deuterated substrates. In order to probe the reaction catalyzed by the cyanobacterial Δ9 desaturase and compare it to the eukaryotic desaturases, the desC gene of Spirulina platensis, strain C1 (Arthrospira sp. PCC 9438) was expressed in a desaturase mutant of baker's yeast. Kinetic isotope effects were performed by culturing yeast transformants with deuterated thia-substituted stearic acids. A large kinetic isotope effect was found for the 9 position, in qualitative agreement with results from eukaryotic desaturases.     

固定化培养中氮磷浓度对钝顶螺旋藻生长及其代谢产物和叶绿素荧光参数的影响

该研究以钝顶螺旋藻为实验对象,探究培养基中不同氮浓度(5、15、30、45 mmol·L^-1)和磷浓度(0.5、1.5、3.0、4.5 mmol·L^-1)对固定化生物膜培养模式下螺旋藻生长及其代谢产物和叶绿素荧光参数的影响,为生产实际中规模化高效培养螺旋藻提供理论依据。结果表明：(1)随着氮、磷浓度的升高,螺旋藻的生物量密度和产率先增高后降低,并在30 mmol·L^-1氮和3.0 mmol·L^-1磷处理时达到最大值;低氮、低磷条件会影响螺旋藻的藻体结构,表现为单细胞藻丝变短,螺旋变少,导致生物膜细胞密度降低。(2)与生物量变化趋势一致,螺旋藻光合色素含量随着培养基中氮、磷浓度的增加呈先升后降的变化趋势,且均在30 mmol·L^-1氮或者3.0 mmol·L^-1磷条件下达到最大值。(3)培养基中磷浓度固定时,螺旋藻的PSⅡ反应中心最大光能转化效率(F_v/F_m)和电子传递速率(ETR)随着氮浓度增加而增大,氮的缺乏会...     

Purification, crystallization and preliminary crystallographic analysis of CYP 195A2, a P450 enzyme from Rhodopseudomonas palustris

Cytochrome P450 monooxygenases are a superfamily of heme-thiolate proteins involved in the metabolism of a wide variety of endogenous and xenobiotic compounds. The P450 enzyme CYP195A2 from Rhodopseudomonas palustris CGA009, a metabolically versatile bacterium, was overproduced in E. coli and purified. Two distinct crystal forms were obtained under separately optimized conditions by the hanging-drop vapor-diffusion method. Native data sets extending to resolutions of 2.3 A and 2.8 A have been collected and processed in space groups P222 and C2221 respectively.     

High temperature induced alterations in energy transfer in phycobilisomes of the cyanobacterium Spirulina platensis

Exposure of intact cells of Spirulina to high temperature (HT) stress (40–60 °C) caused decrease in absorption spectrum and fluorescence emission spectrum. Low temperature emission spectra were altered at phycocyanin (PC) level. Room and low temperature emission spectra of intact phycobilisomes showed that PC was the main target in this cyanobacterium for the altered energy transfer under HT.This revised version was published online in March 2005 with corrections to the page numbers.