We have been studying enzymes that hydrolyze ²-glycosides in the insect midgut for several years and this work intend to shed light in three aspects of this subject.²-galactosidases: We found ²-galactosidases that are activated by hydrophobic substances. Our hypothesis is that these enzymes have galactolipids present in leaves as natural substrates and this activation is important to set them active in the vicinity of the membrane in which these substrates are found. We intend to clone the corresponding cDNA and express a ²-galactosidase from Tenebrio molitor, studying their specificity and activation parameters.²-fructosidases: These enzymes have only been described in Lepidoptera and were not well studied. We intend to use the Spodoptera frugiperda enzyme to determine its substrate specificity, produce site directed mutants to study the modulation of catalytic group pKas and describe their secretion by immunocytolocation. Searching in databases we found sequence highly similar to Lepidoptera ²-fructosidase in Apis mellifera. The presence of the enzyme and the site of its mRNA expression will be verified in A. mellifera.²-glucosidases: We compared kinetically several of these enzymes and we intend to verify if such comparisons can be reproduced using structural data, which will be obtained by molecular modeling and docking . In order to be sure about the sequences of some of them, two enzymes of D. saccharalis will be expressed and have its kinetic parameters determined. One of these enzymes apparently has high activity on O- and S-glycosides. If this result can be confirmed, this property will be studied in greater details.
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