High pressure disruption: a two-step treatment for selective extraction of intracellular components from the microalga Porphyridium cruentum

The microalga Porphyridium cruentum is known to produce many components of interest. One of them is B-Phycoerythrin (B-PE), a water-soluble intracellular pigment used as an immunofluorescent probe. Current methods to extract this molecule involve total cell disruption and lead to a mix of all the water-soluble components. Subsequently, the pigment purification is very complex. An alternative approach to extract B-PE selectively and thus simplify the purification procedure has been developed using a high-pressure cell disrupter. Different pressures (from 27 to 270 MPa), extracting mediums (distilled water and original microalgae culture medium), and numbers of passages (1 to 3) have been tested. Proteins are selectively more extracted than B-PE at low pressure in original medium. It is thus possible to remove part of the intracellular proteins in a first step and then recover enriched B-Phycoerythrin fraction at higher pressure in distilled water.     

Evaluation of the protein quality of Porphyridium cruentum

The amino acid profile of the red microalga Porphyridium cruentum and its protein extract have been determined in order to assess the nutritional quality of this biomass for human consumption. Total protein determined by elemental analysis represented 56 % of its dry weight. Hydro-soluble proteins extracted at pH 12 and 40 °C were analysed by the Lowry method giving 47 %, which represented 84 % of total protein per dry weight. The amino acid sequence of the biomass and the protein extract was composed of a set of essential (39 % for the former and 37 % for the latter) and non-essential amino acids (61 % for the former and 63 % for the latter) that compares favourably with the standard protein/amino acid requirements proposed by Food and Agricultural Organisation and World Health Organisation.     

Effects of nitrogen source and irradiance on <Emphasis Type="Italic">Porphyridium cruentum</Emphasis>

We determined the effects of two nitrogen sources (ammonium and nitrate) and two irradiance levels (50 and 200 μmol photons m^?2 s^?1) on the growth rate, cell size, proximate composition, pigment content, and photosynthesis of the unicellular red alga, Porphyridium cruentum. Irradiance significantly affects growth rate, as well as carbohydrate, protein, and phycoerythrin content. Nitrogen form significantly affects cell size, total dry weight, organic dry weight, ash content, carotene content, phycocyanin content, allophycocyanin content, maximum relative electron transport rate (rETRm), and photosynthetic efficiency (α). However, the irradiance and nitrogen source had significantly interaction with the content of lipids and chlorophyll a content, relative electron transport rate (rETR), and irradiance of saturation (I_k). These findings demonstrate that irradiance and nitrogen source influence the metabolism of P. cruentum and that the combination of these two variables induces the production of chemical products for biotechnological, aquaculture, and nutraceutical industry.     

Method for B-phycoerythrin purification from Porphyridium cruentum

Summary Porphyridium cruentum extract was treated with rivanol for the precipitation and elimination of the polysaccharide typical for this alga, while all phycobiliproteins remained solubilized. After their precipitation with ammonium sulphate, B-phycoerythrin was differentially separated from the other phycobiliproteins, and rivanol was removed by Sephadex G-25 gel filtration. The purity of B-phycoerythrin was proved.Abbreviations B-PE B-phycoerythrin - b-PE b-phycoerythrin - R-PC R-phycocyanin - APC allophycocyanin - PBP phycobiliproteins     

Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors

Additional evidence about the influence of the cell wall physical and chemical characteristics on protein extractability was determined by calculating the conversion factors of five different microalgae known to have different cell wall composition, and their protein extracts. The conversion factors obtained for crude rigid cell walled Chlorella vulgaris, Nannochloropsis oculata and Haematococcus pluvialis were 6.35, 6.28 and 6.25, respectively, but for their protein extracts the values were lower with 5.96, 5.86 and 5.63. On the other hand, conversion factor obtained for fragile cell walled microalgae Porphyridium cruentum and Athrospira platensis was 6.35 for the former and 6.27 for the latter, with no significant difference for their protein extract with 6.34 for the former and 6.21 for the latter. In addition, the highest hydro-soluble protein percentage recovered from total protein was for P. cruentum 80.3 % and A. platensis 69.5 % but lower for C. vulgaris with 43.3 %, N. oculata with 33.3 % and H. pluvialis with 27.5 %. The study spotted the light on the influence of the cell wall on evaluating the conversion factor and protein extractability. In addition, it showed the necessity of finding the conversion factor every time accurate protein quantification is required, and proved that there is not a universal conversion factor that can be recommended.     

PHYSIOLOGICAL CHARACTERISTICS OF PHOTOSYNTHESIS IN PORPHYRIDIUM CRUENTUM: EVIDENCE FOR BICARBONATE TRANSPORT IN A UNICELLULAR RED ALGA1

Various physiological characteristics of photosynthesis in the unicellular red alga Porphyridium cruentum Naegeli have been investigated. The rate of photosynthesis was optimal at 25° C and pH 7.5 and was not inhibited by 21% oxygen over a temperature range of 5 to 35° C. Kinetics of whole cell photosynthesis as a function of substrate concentration gave a K_1/2, (CO₂) of 0.3 μM. CO₂ compensation point, measured in a closed system at pH 7.5, was a constant 6.7 m?L · L^?1 over the temperature range 15 to 30° C and was unaffected by O₂ concentration. Whole cell photosynthesis, measured in a closed system at alkaline pH, showed that the rates of oxygen evolution were greatly in excess of the rate of CO₂ supply from the spontaneous dehydration of HCO₃^? in the medium. This indicates that bicarbonate is utilized by the cell to support this photosynthetic rate. These physiological characteristics of Porphyridium cruentum are consistent with the hypothesis that this alga transports bicarbonate across the plasmalemma.     

A novel cold-adapted β-galactosidase isolated from Halomonas sp. S62: gene cloning, purification and enzymatic characterization

A novel 1,170 bp β-galactosidase gene sequence from Halomonas sp. S62 (BGalH) was identified through whole genome sequencing and was submitted to GenBank (Accession No. JQ337961). The BGalH gene was heterologously expressed in Escherichia coli BL21(DE3) cells, and the enzymatic properties of recombinant BGalH were studied. According to the polyacrylamide gel electrophoresis results and the sequence alignment analysis, BGalH is a dimeric protein and cannot be classified into one of the known β-galactosidase families (GH1, GH2, GH35, GH42). The optimal pH and temperature were determined to be 7.0 and 45 °C, respectively; the K _m and K _cat were 2.9 mM and 390.3 s^?1, respectively, for the reaction with the substrate ortho-nitrophenyl-β-d-galactopyranoside. At 0–20 °C, BGalH exhibited 50–70 % activity relative to its activity under the optimal conditions. BGalH was stable over a wide range of pHs (6.0–8.5) after a 1 h incubation (>93 % relative activity) and was thermostable at 50 °C and below (>60 % relative activity). The enzyme hydrolyzes lactose completely in milk over 24 h at 7 °C. The characteristics of this novel β-galactosidase suggest that BGalH may be a good candidate for medical researches and food industry applications.     

Optimization of renewal regime for improvement of polysaccharides production from Porphyridium cruentum by uniform design

To probe the effects of renewal regime on the production of polysaccharides, Porphyridium cruentum was cultured semi-continuously in flat plate photobioreactor. Uniform design was used to optimize renewal conditions. Quadratic mathematic models related to productivity, total recovery yield of biomass and polysaccharides were set up to clarify the influence of individual factors and their interactions. According to the mathematic models, the optimal semi-continuous condition for total yield of polysaccharide was NaNO₃ 3.5 g/L, renewal rate 27%, renewal period 2.91 days. The optimal condition for polysaccharide output rate was NaNO₃ 0.5 g/L, renewal rate 5%, renewal period 7 days. With the optimal renewal regime, the maximal total recovery yields of polysaccharide achieved at 29.4 g, which was 1.57 times higher than that of batch cultivation. The maximum output rate of polysaccharide was 68.64 mg/L per day, which was 2.02 times higher than previous reported data.     

三种紫球藻亲缘关系RAPD分析

利用RAPD技术对铜绿紫球藻（Porphyridium aerugineum 755）,淡色紫球藻（Porphyridium purpureum 806）和紫球藻（Porphyridium cruentum）的亲缘关系进行分析。从50个随机引物中筛选出25个种间多态性较强,重复性较好的引物。检测到233个位点。在233个条带中有186个多态性位点。多态位点比率为79.73%,平均每个引物扩增9个条带,多态性条带7个。聚类分析结果表明：铜绿紫球藻、淡色紫球藻和紫球藻之间的平均遗传距离为0.455,其中铜绿紫球藻和淡色紫球藻之间的遗传距离为0.413,铜绿紫球藻和紫球藻之间的遗传距离为0.556,淡色紫球藻和紫球藻之间的遗传距离最小为0.396。所以由RAPD试验的分析结果可以得出:三种紫球藻为独立的种, 其中淡色紫球藻和紫球藻是亲缘关系较近的两个不同种。紫球藻属种间个体DNA多态性比较丰富,因此利用RAPD技术可以从DNA水平上检测紫球藻属种间差异。     

Comparative properties of ferredoxins from a marine and freshwater species of Porphyridium

Ferredoxins were isolated from the freshwater red alga Porphyridium aerugineut, and from Porphyridium cruentum, a related marine species. A sin     

The quaternary structure of a unique phycobiliprotein: B-phycoerythrin from Porphyridium cruentum

B-phycoerythrin, from the unicellular red alga Porphyridium cruentum, was crystallized in the rhombohedral space group R3 with a=111.0Å and α=116.8° or A=B=189.1Å and C=60.1Å and γ=120°. Density measurements on the crystals indicate that the hexagonal unit cell can acconmodate three cylindrical molecules, 109Å in diameter and 60Å in height, each of approximately 275,000 daltons. The crystallographic symmetry of the unit cell requires at least 3-fold symmetry for the particle. However, the particle stoichiometry has been reported as (αβ)₆γ and this composition is also supported by SDS gel electrophoresis on the crystalline protein. These results are discussed in light of preliminary model calculations on the quaternary structure of B-phycoerythrin.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of Porphyridium cruentum which had been frozen to ?196 °C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at ?196 °C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Light-induced energetic decoupling as a mechanism for phycobilisome-related energy dissipation in red algae: a single molecule study

Background

Photosynthetic organisms have developed multiple protective mechanisms to prevent photodamage in vivo under high-light conditions. Cyanobacteria and red algae use phycobilisomes (PBsomes) as their major light-harvesting antennae complexes. The orange carotenoid protein in some cyanobacteria has been demonstrated to play roles in the photoprotective mechanism. The PBsome-itself-related energy dissipation mechanism is still unclear.

Methodology/Principal Findings

Here, single-molecule spectroscopy is applied for the first time on the PBsomes of red alga Porphyridium cruentum, to detect the fluorescence emissions of phycoerythrins (PE) and PBsome core complex simultaneously, and the real-time detection could greatly characterize the fluorescence dynamics of individual PBsomes in response to intense light.

Conclusions/Significance

Our data revealed that strong green-light can induce the fluorescence decrease of PBsome, as well as the fluorescence increase of PE at the first stage of photobleaching. It strongly indicated an energetic decoupling occurring between PE and its neighbor. The fluorescence of PE was subsequently observed to be decreased, showing that PE was photobleached when energy transfer in the PBsomes was disrupted. In contrast, the energetic decoupling was not observed in either the PBsomes fixed with glutaraldehyde, or the mutant PBsomes lacking B-PE and remaining b-PE. It was concluded that the energetic decoupling of the PBsomes occurs at the specific association between B-PE and b-PE within the PBsome rod. Assuming that the same process occurs also at the much lower physiological light intensities, such a decoupling process is proposed to be a strategy corresponding to PBsomes to prevent photodamage of the photosynthetic reaction centers. Finally, a novel photoprotective role of γ-subunit-containing PE in red algae was discussed.     

Effective Absorption Cross-Sections in Porphyridium cruentum: Implications for Energy Transfer between Phycobilisomes and Photosystem II Reaction Centers

Effective absorption cross-sections for O₂ production by Porphyridium cruentum were measured at 546 and 596 nanometers. Although all photosystem II reaction centers are energetically coupled to phycobilisomes, any single phycobilisome acts as antenna for several photosystem II reaction centers. The cross-section measured in state I was 50% larger than that measured in state II.     

Preparation of different molecular weight polysaccharides from Porphyridium cruentum and their antioxidant activities

Hermetical microwave was used to degrade Porphyridium cruentum polysaccharides from 2918 to 256.2, 60.66 and 6.55 kDa. The antioxidant properties of different molecular weight polysaccharides were evaluated by determining the scavenging ability of free radicals, inhibitory effects on lipid peroxidation in liver homogenates and hemolysis of mouse erythrocytes. Analysis of physicochemical properties confirmed that microwave degradation might not markedly change the chemical components of the polysaccharides. High-molecular-weight polysaccharides from P. cruentum had no obvious antioxidant activity, but low-molecular-weight fragments after degradation exerted an inhibitory effect on oxidative damage. The 6.55-kDa fragment had stronger antioxidant activity than the 60.66 and 256-kDa fragments.     

Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

Cloning and expression of A. oryzae β-glucosidase in Pichia pastoris

A β-glucosidase gene (bgl) from Aspergillus oryzae GIF-10 was cloned, sequenced and expressed. Its full-length DNA sequence was 2,903 bp and included three introns. The full-length cDNA sequence contained an open reading frame of 2,586 nucleotides, encoding 862 amino acids with a potential secretion signal. The A. oryzae GIF-10 bgl was functionally expressed in Pichia pastoris. After 7-day induction, protein yield reached 321 mg/mL. Using salicin as the substrate, the specific activity of the purified enzyme reached 215 U/mg. The purified recombinant β-glucosidase was a 110-kDa glycoprotein with optimum catalytic activity at pH 5.0 and 50 °C. The enzyme was stable between 20 and 60 °C, and retained 65 % of its activity after being held at 60 °C for 30 min. The recombinant β-glucosidase was relatively stable in a broad range of pHs, from 4.0 to 6.5. It showed broad specific activity, hydrolyzing a range of (1-4)-β-diglycosides and (1-4)-α-diglycosides, and Mn²⁺ stimulated its activity significantly.     

Mixotrophic production of phycoerythrin and exopolysaccharide by the microalga Porphyridium cruentum

The ability of the unicellular rhodophyte Porphyridium cruentum to grow mixotrophically on the soluble fraction of Solarium tuberosum meal was tested. At the beginning of stationary phase Porphyridium cruentum produced 7 μg ml⁻¹ of phycoerythrin and 129 μg ml⁻¹ of total soluble exopolysaccharide when cultured autotrophically. When cultured mixotrophically with the soluble fraction of Solanum tuberosum meal, the productivity increased to 10 μg ml⁻¹ of phycoerythrin and 330 μg ml⁻¹ of total soluble exopolysaccharide. When the soluble fraction of S. tuberosum meal was supplied together with nitrate and phosphate, the productivity of phycoerythrin increased to 21 μg ml⁻¹ while the production of total soluble exopolysaccharide decreased to 195 μg ml⁻¹. Results demonstrate that the soluble fraction of S. tuberosum meal can be used as substrate for the production of phycoerythrin and exopolysaccharide by P. cruentum improving the results obtained with the autotrophic culture medium.     

Optimization of heterologous expression of the phytase (PPHY) of Pichia anomala in P. pastoris and its applicability in fractionating allergenic glycinin from soy protein

The phytase (PPHY) of Pichia anomala has the requisite properties of thermostability and acidstability, broad substrate spectrum, and protease insensitivity, which make it a suitable candidate as a feed and food additive. The 1,389-bp PPHY gene was amplified from P. anomala genomic DNA, cloned in pPICZαA, and expressed extracellularly in P. pastoris X33. Three copies of PPHY have been detected integrated into the chromosomal DNA of the recombinant P. pastoris. The size exclusion chromatography followed by electrophoresis of the pure rPPHY confirmed that this is a homohexameric glycoprotein of ~420 kDa with a 24.3 % portion as N-linked glycans. The temperature and pH optima of rPPHY are 60 °C and 4.0, similar to the endogenous enzyme. The kinetic characteristics K _m, V _max, K _cat, and K _cat/K _m of rPPHY are 0.2 ± 0.03 mM, 78.2 ± 1.43 nmol mg^?1 s^?1, 65,655 ± 10.92 s^?1, and 328.3 ± 3.12 μM^?1 s^?1, respectively. The optimization of medium components led to a 21.8-fold improvement in rPPHY production over the endogenous yeast. The rPPHY titer attained in shake flasks could also be sustained in the laboratory fermenter. The rPPHY accounts for 57.1 % of the total secreted protein into the medium. The enzyme has been found useful in fractionating allergenic protein glycinin from soya protein besides dephytinization.     

Indoor mass cultivation of red alga Porphyridium cruentum in different types of bioreactors: effect of scale-up and vessel shape

The present study describes scale-up of Porphyridium cruentum cultures in different types of enclosed bioreactors. Special consideration for the purpose was the comparative performance of these photobioreactors relevant to growth and polysaccharide production, which have a significance in the commercial exploitation of the microalga. The custom-built flat-sided photobioreactor with higher exposed surface area to volume ratio was found to be the best system for the cultivation of P. cruentum.