Immobilisation of engineered molecules on electrodes and optical surfaces,

ABSTRACT Monolayers of genetically modified proteins with an hexahistidine tag, (His)6, were obtained by using a Ni -NTA chelator synthesized on gold sputtered surfaces (via sulphide bonds), or on gold and graphite (via sililating agents) working electrodes of screen-printed devices. Two kinds of proteins were produced and purified for this study: a) a recombinant antibody, derived from the 'single chain Fv' ( scFv) format; b) a photosystem II (PSII) core complex isolated from the mutant strain CP43-H of the thermophilic cyanobacterium Synechococcus elongatus. An scFv, previously isolated from a synthetic 'phage display' library, was further engineered with an alkaline phosphatase activity genetically added between the carbossi-terminal of the scFvs and the (His)6 to allow direct measurement of immobilisation. Renewable specific binding of (His)6-proteins to gold and graphite surfaces and fast and sensitive electrochemical or optical detection of analytes were obtained. Additionally, "on chip" protein preconcentration was conveniently achieved for biosensing purposes, starting from crude unpurified extracts and avoiding protein purification steps

Non-Photochemical Quenching in Cryptophyte Alga Rhodomonas salina Is Located in Chlorophyll a/c Antennae

Photosynthesis uses light as a source of energy but its excess can result in production of harmful oxygen radicals. To avoid any resulting damage, phototrophic organisms can employ a process known as non-photochemical quenching (NPQ), where excess light energy is safely dissipated as heat. The mechanism(s) of NPQ vary among different phototrophs. Here, we describe a new type of NPQ in the organism Rhodomonas salina, an alga belonging to the cryptophytes, part of the chromalveolate supergroup. Cryptophytes are exceptional among photosynthetic chromalveolates as they use both chlorophyll a/c proteins and phycobiliproteins for light harvesting. All our data demonstrates that NPQ in cryptophytes differs significantly from other chromalveolates – e.g. diatoms and it is also unique in comparison to NPQ in green algae and in higher plants: (1) there is no light induced xanthophyll cycle; (2) NPQ resembles the fast and flexible energetic quenching (qE) of higher plants, including its fast recovery; (3) a direct antennae protonation is involved in NPQ, similar to that found in higher plants. Further, fluorescence spectroscopy and biochemical characterization of isolated photosynthetic complexes suggest that NPQ in R. salina occurs in the chlorophyll a/c antennae but not in phycobiliproteins. All these results demonstrate that NPQ in cryptophytes represents a novel class of effective and flexible non-photochemical quenching

Interplay between photochemical activities and pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to red stage

9th International Conference on Applied Algology -- MAY 26-30, 2002 -- AGUADULCE, SPAINWOS: 000183045600007The transfer of laboratory cultures of H. pluvialis to high irradiance outdoors caused a substantial decline in the maximum quantum yield of photosystem II (PSII), from 0.65 in the morning to 0.45 at midday, as measured by the ratio of variable to maximum fluorescence yields (F(v)/F(m)), and a steep rise in non-photochemical quenching (NPQ). Chlorophyll fluorescence induction curves of morning samples showed a clear I-step, reflecting a certain PSII heterogeneity. Single turnover flash measurements on samples taken from the outdoor photobioreactor in the middle of day showed an increase in the reoxidation time constant of the reduced plastoquinone Q(A)(-), i.e., the time required for electron transfer from the primary plastoquinone acceptor of PSII Q(A)(-) to the secondary plastoquinone acceptor Q(B). Photosynthesis rates were almost constant during the day. Along with the increase in non-photochemical quenching, there was a slight increase in zeaxanthin and antheraxanthin contents and decrease in violaxanthin, showing the presence of an operative xanthophyll cycle in this microalga. A marked increase of secondary carotenoids was found at the end of the first day of exposure to sunlight, mainly astaxanthin monoester, which reached 15.5% of the total carotenoid content. Though cells turned reddish during the second day, the decline in the fluorescence parameter F(v)/F(m) in the middle of the day was less than during the first day, and there was no further increase in the value for NPQ. Similar behaviour was observed during the third day when the culture was fully red. After four days of exposure to sunlight, the dry weight reached 800 mg L(-1) and the concentration of secondary carotenoids (81% astaxanthin monoester) reached 4.4% dry weight.Int Soc Appl Phyco

Synergistic effect of UV radiation and nutrient limitation on chlorella fusca (Chlorophyta) cultures grown in outdoor cylindrical photobioreactors

This study assessed the interactive effects of UVR and nutrient depletion on Chlorella fusca cultures on the production and accumulation of particular biomolecules. To accomplish this, algae were grown for 5 d in outdoor thin-layer cascade cultivators under 3 nutrient treatments (full nutrients, -N and -S) and then transferred to outdoor cylindrical photobiore-actors for another 5 d. Cultures were then exposed to full solar radiation (PAB) and decreased UVR. During the last 5 d, bio-optical properties, photosynthetic activity, pigments, biochemical composition and oxidative stress were assessed. Initially, nutrient depletion caused changes in productivity and cell number in a manner that affected biochemical composition. After 3 d, the percentage of lipids in the cultures under N deprivation reached values appropriate for being used as feed or food additives or for energy applications (35% of lipid content), regardless of the light conditions. A longer exposure (5 d) resulted in interactive effects of light and nutrient conditions. Specifically, PAB increased lipid content in all cases (1.3- to 2.3-fold), but particularly under S deprivation. Longer exposure to PAB also increased oxidative stress in UVR and nutrient-limited treatments (-N and -S). These results showed that the benefits expected from nutrient depletion (increase in biomolecule content e.g. lipids, carbohydrates and pigments) were modulated by the negative effects of algal UVR acclimation costs

Reversible immobilization of engineered molecules by Ni-NTA chelators

Abstract Electrochemical synthesis of nickel-nitrilotriacetic acid (Ni-NTA) chelators, for subsequent immobilization of (His) 6 -tagged proteins (Photosystem II (PSII) as model molecule), on Au or Au-graphite electrodes is compared to chemical synthesis. Results show: (i) higher Ni-NTA surface density, (ii) shorter treatment time (1 -12 min vs. 16 h normally needed for self-assembled monolayer (SAM)), (iii) possibility of addressing the chelator to only one Au electrode, in a sensor A-array.