engleski

"Structure is function": A contribution to understanding enzymatic activity of bacterial lipases.

The results to be presented are related to an interdisciplinary approach in the research of bacterial lipases. An investigation has been focused on: known lipase from Pseudomonas cepacia (PCL) and a novel extracellular lipase from Streptomyces rimosus R6-554W. To understand the mechanism of their activity and to use them efficiently in further research and application, the 3-D structure has to be known. Therefore, we started our research with the known enzyme PCL to test various in-house prepared substrates and inhibitors with a goal to use lipases as catalyst in organic synthesis to obtain enantio-pure products. To resolve the faster reacting enantiomers and to find out their mode of binding into the enzyme active site, molecular modelling was extensively used. The enzyme-inhibitor complex was prepared and its crystal structure determined. The experimental results were compared with computer modelling and good agreement was achieved. The novel lipase from Streptomyces rimosus R6-554W was isolated and biochemically characterized. It is a monomeric, basic protein, active toward triolein and p-nitrophenyl esters, with the preference for those with medium size acyl chain length. Interfacial activation was observed with p-nitrophenyl butyrate as a substrate. The lipase is the most active at 50-60 C in alkaline conditions around pH 9-10, with p-nitrophenyl palmitate as a substrate. It showed high stability at a broad pH range of 4-10 and was fairly thermostable. To obtain the amount of protein for more extended studies, lipase was cloned and sequenced. Comparison to known streptomycete lipases characterized so far, reveals significant differences biochemically and genetically suggesting higher variability of lipases than expected in this bacterial genus. Crystallization experiments of novel lipase are in due course.

lipase; structure; Streptomyces rimosus; Pseudomonas cepacia

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