Characterization of thermophilic lipolytic enzymesSearching and study of microorganisms and enzymes with biotechnological interest from galician hot springs

Resumo

Galicia (north-western Spain) is a region with a valuable richness in hot springs with a broad diversity of interesting hyperthermophile microorganisms, for instance, as for potential production of enzymes with biotechnological purposes. In spite of it, basically only physicochemical parameters of those springs have been studied. Among all, two of them share the particularity of having the highest upwelling temperature (66 ºC). These hot springs are named As Burgas (Ourense) and Río Caldo (Lobios, Ourense). The last one, in addition, has a pH of 9. As an example of interesting thermophilic enzymes as for biotechnological aims, esterases are noteworthy in fields such as pharmaceutical industry (chiral compounds isolating), energy production (biofuel) or food industry (dairy or flavours synthesis). These biocatalysts are normally an excellent alternative to other methodologies when scaling up a process to industry level. In our research group, a working line focused on finding thermophile microorganisms producing enzymes with biotechnological interest has been initiated. Wild type microorganisms from the genus Thermus were cultured and esterases from them were identified and purified; this process was patented. Furthermore, in previous projects collaborating with the University of A Coruña, procedures for the expression of these enzymes in mesophiles and their purification were performed. Paying attention to this experience, the main objectives of the thesis project will be, firstly, producing, purifying and characterizing new thermophilic lipolytic enzymes and, secondly, due to the high arsenic concentration in some of the springs, studying the possible involvement of microorganisms (and their enzymes) from these water samples in bioremediation processes related to arsenic oxidation (interesting as for consumption water quality). Objectives: 1. Use of Molecular Microbiological Ecology techniques for carrying out an analysis of the hot springs microbial community biodiversity, comparing seasonal diversity profiles and identifying microorganisms of interest. It will also be performed a complete chemical study of water samples, focusing on all those factors that could have influence on the biodiversity and on richness in genes with biotechnological potential. So, other springs could be identified for new prospections. 2. Production and purification of new thermophilic lipolityc enzymes. The initial cultures will be done in flasks. Cellular density, protein concentration and activity will be measured through conventional methods. Esterases with high levels of activity and stability could be produced in a 5 l reactor, and at a semi-industrial scale (50 l) with bioreactors located at the CITI (Center of Resarch, Transfer and Innovation) building. 3. Kinetic and molecular characterization of purified lipolityc enzymes. Experimental design for evaluating pH and temperature optima and theirs effects on activity and stability will be performed. The effect of solvents and detergents will be studied in the presence of several chemicals. Activity will be measured with p-nitrophenyl esters and triglycerides. The stability could also be studied through fluorescence techniques, and structural analysis through circular dichroism. 4. Isolation of thermophile microorganisms able to grow on arsenite, interesting as for bioremediation processes (oxidation of AsIII to AsV, being the last one easly recovered from consumption waters). Once tested the best conditions for growth of isolates and their resistance to AsIII, confirmation of their ability to oxidize could be performed through different procedures, such as spectrometry, qualitative techniques or molecular biology.