Microbial lipases with interest in biotechnology and infectious diseases: isolation, characterization and inhibition by natural substances

Lipases are carboxylic ester hydrolases which act on acylglycerols to liberate fatty acids and glycerol. These enzymes are currently attracting an enormous attention because they are among the most versatile and widely used enzymes in biotechnological applications and due to their unique properties. Moreover, these enzymes and their inhibitors have a high pharmacological interest because some microbial lipases can act as virulence factors in several infectious diseases. Therefore, the general objective of the present work was the isolation, cloning, characterization and inhibition by natural substances of microbial lipases with interest in biotechnology and infectious diseases.The first chapter was focused on the isolation and characterization of new Bacillus lipases to evaluate their biotechnological potential. The lipolytic system of several very active strains with an unknown lipolytic system was analyzed, and then, the lipases LipA from Bacillus megaterium CECT 370 and LipA from Bacillus sp. BP-6 were isolated, cloned and characterized. Both enzymes are family I.4 carboxylesterases closely related to Bacillus subtilis LipB, and have a high biotechnological potential due to their high stability and due to their molecular and biochemical properties.Chapter 2 consisted in the isolation of 724 microorganisms from soil samples collected from a subtropical forest of Puerto Iguazú (Argentina). Among them, 449 showed one or more of the biotechnologically-interesting enzymatic activities "true lipase", carboxylesterase, cellulase, xylanase and pectinase. CR-53 and CR-179, two very active isolates, were subsequently identified as two strains closely related to the species Rhodococcus erythropolis and Bacillus subtilis, respectively. Further analysis revealed that strain CR-53 produces a cell-bound carboxylesterase of 60 kDa, one of the first lipases known in the genus Rhodococcus, whereas strain CR-179 possesses a lipolytic system related to that of other Bacillus.Chapter 3 was focused in the development of a new, fast, simple and more sensitive colorimetric microassay with a low cost and suitable for high-throughput analysis of purified or non-purified lipolytic enzymes. The assay was subsequently used to evaluate the effect of several saturated fatty acids on five cell-bound or secreted (Paeni)Bacillus lipases. These lipids inhibited all the enzymes analyzed, although secreted lipases were activated by low concentrations of these compounds. Activation of microbial lipases by fatty acids is a phenomenon detected here for the first time, and could be related to the properties and biological function of these secreted enzymes.Chapter 4 consisted in the analysis of the inhibitory effect of several natural substances (saponins, flavonoids and alkaloids) on the model lipase from Candida rugosa (CRL) to evaluate their potential as antilipase drugs. beta-aescin, digitonin, kaempferol and (±)-catechin were selected as the best candidates for the treatment or prevention of lipase-related diseases due to the strong inhibition they produced on CRL, as well as due to their other beneficial effects and their low toxicity.The aim of chapter 5 was to isolate, clone, characterize and evaluate the inhibition of lipases from the pathogens Propionibacterium acnes and Helicobacter pylori. The analysis of GehA from Propionibacterium acnes P-37, a lipase considered as a major etiological agent in the pathogenesis of acne, revealed that this enzyme is very adapted to the skin conditions. EstV (HP0739), a family V carboxylesterase which was identified by analyzing Helicobacter pylori 26695 proteome, is the first lipase from this pathogen that has been cloned, purified and characterized. The evaluation of the effect of several natural substances on GehA and EstV revealed that beta-aescin, glycyrrhizic acid, (±)-catechin and kaempferol are promising candidates for the treatment of acne and/or ulcer due to their strong inhibitory activity on these lipases, as well as due to their other anctiacne or antiulcer effects and their low toxicity