Hydrolases constitute a class of enzymes widely distributed in nature from bacteria to higher eukaryotes. The halotolerance of many enzymes derived from halophilic bacteria can be exploited wherever enzymatic transformations are required to function under physical and chemical conditions, such as in the presence of organic solvents and extremes in temperature and salt content. In recent years, different screening programs have been performed in saline habitats in order to isolate and characterize novel enzymatic activities with different properties to those of conventional enzymes. Several halophilic hydrolases have been described, including amylases, lipases and proteases, and then used for biotechnological applications. Moreover, the discovery of biopolymer-degrading enzymes offers a new solution for the treatment of oilfield waste, where high temperature and salinity are typically found, while providing valuable information about heterotrophic processes in saline environments. In this work, we describe the results obtained in different screening programs specially focused on the diversity of halophiles showing hydrolytic activities in saline and hypersaline habitats, including the description of enzymes with special biochemical properties. The intracellular lipolytic enzyme LipBL, produced by the moderately halophilic bacterium Marinobacter lipolyticus, showed advantages over other lipases, being an enzyme active over a wide range of pH values and temperatures. The immobilized LipBL derivatives obtained and tested in regio- and enantioselective reactions, showed an excellent behavior in the production of free polyunsaturated fatty acids (PUFAs). On the other hand, the extremely halophilic bacterium, Salicola marasensis sp. IC10 showing lipase and protease activities, was studied for its ability to produce promising enzymes in terms of its resistance to temperature and salinity.

BackgroundAmong extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.Methods and FindingsA lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.ConclusionsIn this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.

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Oxygen diffusion in soilless culture systems in Mediterranean climate conditions is often quite low, especially in summer. This fact is seldom taken into account even though it could lead to plant development constraints, as for instance scarce root development or certain nutrient uptake deficits. A study of a rose crop in a soilless recirculating system with oxifertigation and nightly oxygen supply has been carried out to determine whether there is a cause-effect relationship between the production and growth increase in soilless culture systems which have been supplied with oxygen and the plant metabolism optimization due to the absorption of different nutrients. One and two years old cv. Red Corvette plants were planted in containers with two substrates (perlite B6 and A13). They were grown with or without diurnal oxifertirrigation and with or without night oxygenation in a 2x2x2 factorial experiment. A robot automatically controlled fertirrigation, lixiviated disinfection, nutrient solution recomposition from lixiviates, watering and fertilize. Nutrient solution was continuously held with oxygen over saturation. Night oxygen supply was done directly into the substrate by sequent pulsations. Nutrient content on leaves and petioles were determined using: ion-sensible electrode (nitrogen); Bray-Kurtz colorimetric method following by a spectrophotometer (λ=660nm) (phosphorus); flame photometry (potassium) and atomic spectroscopy analysis (iron, calcium and magnesium). Differences in foliar mineral contents were observed between two produced fluxes: Moreover, calcium and magnesium leaf content was also higher in plants grown in A13 perlite substrate. Foliar nutrient's content in oxifertigation treatments were only different in potassium and calcium. They were significative upper in diurnal oxifertigation plants. Significative differences did not appear either in the rest of elements or night oxygen supply conditions. Calcium foliar content increase with diurnal oxifertigation treatment ant this happens with any of the nocturnal treatments. Increase in potassium foliar content in oxifertigation treatment is bigger with nocturnal oxygen supply in B6 substrate, possible because of its long woody life with important nutrient's stocks and its harvest time (earlier phases of flower development).

Marinobacter lipolyticus strain SM19, isolated from saline soil in Spain, is a moderately halophilic bacterium belonging to the class Gammaproteobacteria. Here, we report the draft genome sequence of this strain, which consists of a 4.0-Mb chromosome and which is able to produce the halophilic enzyme lipase LipBL.