Immobolized Staphylococcus xylosus lipase-catalysed synthesis of ricinoleic acid esters

peer reviewe

A Novel Thermostable and Alkaline Protease Produced from Bacillus stearothermophilus Isolated from Olive Oil Mill Sols Suitable to Industrial Biotechnology

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications

Structure and Binding of the C‑Terminal Segment of R9AP to Lipid Monolayers

Phototransduction cascade takes place in disc membranes of photoreceptor cells. Following its activation by light, rhodopsin activates the G-protein transducin causing the dissociation of its GTP-bound α-subunit, which in turn activates phosphodiesterase 6 (PDE6) leading to the hyperpolarization of photoreceptor cells. PDE6 must then be inactivated to return to the dark state. This is achieved by a protein complex which is presumably anchored to photoreceptor disc membranes by means of the transmembrane C-terminal segment of RGS9-1-Anchor Protein (R9AP). Information on the secondary structure and membrane binding properties of the C-terminal segment of R9AP is not yet available to further support its role in the membrane anchoring of this protein. In the present study, circular dichroism and infrared spectroscopy measurements have allowed us to determine that the C-terminal segment of human and bovine R9AP adopts an α-helical structure in solution. Moreover, this C-terminal segment has shown affinity for most of the phospholipids typical of photoreceptor membranes. In fact, the physical state and the type of phospholipid as well as electrostatic interactions influence the binding of the human and bovine peptides to phospholipid monolayers. In addition, these measurements revealed that the human peptide has a high affinity for saturated phosphocholine, which may suggest a possible localization of R9AP in photoreceptor microdomains. Accordingly, infrared spectroscopy measurements have allowed determining that the C-terminal segment of R9AP adopts an ordered α-helical structure in the presence of saturated phospholipid monolayers. Altogether, these data are consistent with the typical α-helical secondary structure and behavior observed for transmembrane segments and with the proposed role of membrane anchoring of the C-terminal segment of human and bovine R9AP

Biodiesel Production by Single and Mixed Immobilized Lipases Using Waste Cooking Oil

Biodiesel is one of the important biofuels as an alternative to petroleum-based diesel fuels. In the current study, enzymatic transesterification reaction was carried out for the production of biodiesel from waste cooking oil (WCO) and experimental conditions were optimized, in order to reach maximum biodiesel yield. Bacillus stearothermophilus and Staphylococcus aureus lipase enzymes were individually immobilized on CaCO3 to be used as environmentally friendly catalysts for biodiesel production. The immobilized lipases exhibited better stability than free ones and were almost fully active after 60 days of storage at 4 °C. A significant biodiesel yield of 97.66 ± 0.57% was achieved without any pre-treatment and at 1:6 oil/methanol molar ratio, 1% of the enzyme mixture (a 1:1 ratio mixture of both lipase), 1% water content, after 24 h at 55 °C reaction temperature. The biocatalysts retained 93% of their initial activities after six cycles. The fuel and chemical properties such as the cloud point, viscosity at 40 °C and density at 15 °C of the produced biodiesel complied with international specifications (EN 14214) and, therefore, were comparable to those of other diesels/biodiesels. Interestingly, the resulting biodiesel revealed a linolenic methyl ester content of 0.55 ± 0.02% and an ester content of 97.7 ± 0.21% which is in good agreement with EN14214 requirements. Overall, using mixed CaCO3-immobilized lipases to obtain an environmentally friendly biodiesel from WCO is a promising and effective alternative for biodiesel production catalysis

Interaction of the Spo20 Membrane-Sensor Motif with Phosphatidic Acid and Other Anionic Lipids, and Influence of the Membrane Environment

<div><p>The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to <i>in vitro</i> and <i>in vivo</i> experiments and allows unbiased comparison between amphipathic helices of different chemistry. <i>In vitro</i>, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.</p></div

Membrane binding properties of the Spo20 bioprobe.

<p>(<b>A</b>) Spo20-GCC (0.75 µM) was incubated with or without PC/PE/Cholesterol liposomes (0.75 mM) containing (mol %) PE (25), cholesterol (25) and supplemented with PA (15), PS (30), or PIP₂ (5). The remaining lipid was PC. Bound proteins were recovered by flotation and analyzed by SDS-polyacrylamide gel electrophoresis using Sypro orange staining. (<b>B, C</b>) Binding of Spo20-GCC to liposomes containing (mol %) PE (25), cholesterol (25) and increasing amounts of PA (circles) or PS (triangles) as indicated. The remaining lipid was PC. A dose response curve for PA with liposomes containing 25 mol % PE, 25 mol % cholesterol and 15 mol % PS is also shown (squares). Membrane partitioning was assessed either by the NBD fluorescence assay (<b>B</b>) or by the liposome flotation assay (<b>C</b>). The data shown for the NBD assay are from two independent experiments. The horizontal dashed line indicates the fluorescence level of the NBD proteins in solution. The vertical bars show the standard deviation of the NBD fluorescence intensity of Spo20-GCC. For the flotation assay, the data shown are from two or three independent experiments with different preparations of liposomes. An typical SDS gel analysis is shown. The dose response curves are shown either as a function of the mol % of PA or PS (left) or as a function of the total amount of negative charges in the membrane (right). (<b>D</b>) Effect of PE on the membrane partitioning of Spo20-GCC as assessed by NBD fluorescence. The liposomes contained (mol %) PS (15), cholesterol (25), PA (0, triangles; 10, squares) and increasing amounts of PE at the expense of PC. All liposomes were prepared by extrusion through 0.2 µm filters.</p

Inverting the Spo20 sequence affects neither its specificity for negatively charged lipids nor its subcellular localization.

<p>(<b>A</b>) Helical wheel representations of the Spo20 sequence and of the corresponding inverted mutant (iSpo20) where the sequence is read from the C- to the N-terminus. (<b>B</b>) Flotation Assays. Binding of iSpo20-GCC to liposomes (0.75 mM lipids; extrusion 0.2 µm) containing (mol %) PE (25), cholesterol (25), PS (0, open symbols; 15, filled symbols) and increasing amounts of PA. The experiment was repeated two or three times with different preparations of liposomes. Data show mean ± S.E of these independent experiments. (<b>C</b>) Confocal microscopy images of RPE1 cells after transfection with Spo20-ACC1-mCherry and iSpo20-ACC2-GFP. xy planes and z projections are shown. Note that the two probes co-localize almost perfectly. Scale bars = 10 µm.</p

Membrane binding properties of the swap Spo20 mutant.

<p>(<b>A</b>) Helical wheel representations of the Spo20 sequence and of the corresponding swap mutant. (<b>B–C</b>) NBD fluorescence assays comparing the membrane partitioning of the [NBD]Spo20-GCC and [NBD]swapSpo20-GCC bioprobes (0.3 µM) to various liposomes (0.35 mM). In (<b>B</b>), the liposomes contained (mol %) PE (25), cholesterol (25), PS (0 or 15) and increasing amounts PA. The remaining lipid was PC. In (<b>C</b>), the liposomes contained (mol %) PC (25), PE (25), cholesterol (25), PA (10) and PS (15) and the partitioning of the constructs was tested at various pH. Data shown are mean ± S.E of 3 independent experiments. All liposomes were prepared by extrusion through 0.2 µm polycarbonate filters.</p