Resumo

Introduction: The use of microbial peptidases has obtained relevance in several industrial segments. Filamentous fungi are a good source for peptidases production due to saprophytic lifestyle and wide biochemical diversity. The enzyme capacity of degrade substrates is an important parameter to determine the enzyme specifity. The aim of this work was to determine the kinetics parameters involved in catalysis of synthetic peptides based on fluorescence peptidase assays. Material and Methods: The assays were performed in spectrofluorometer using synthetic fluorescence resonance energy transfer (FRET) peptides with mutations in amino acids residues in P₁, P₂ and P₃ positions of standard substrate Abz-KLRSSKQ-EDDnp. The excitation and emission wavelength were 320nm and 420nm, respectively. The optimal conditions of temperature (40 ºC) and pH (6.5) were used in all kinetics assays to determine parameters K_M, k_cat e k_cat/K_M. Discussion of results: The highest catalytic efficient 5283 µM^-1 s^-1 was obtained with Arg in substrate P₁ position. The substrate with cistein in same position presented a low interaction with substrate (K_M 20.52 µM). The S₂ subsite demonstrated preference by hydrophobic amino acids because only substrates contained Ile, Pro and Cys in P₂ position were cleaved and presented catalytic efficient 765, 503 and 54.5 µM^-1 s^-1, respectively. The high values of catalytic efficient 3258 and 3106 µM^-1 s^-1 was obtained with basic amino acids residues His and Arg in substrate P₃ position, respectively. In same position, the substrates with acid amino acids residues Glu and Asp demonstrated lower interaction than others substrates and K_M 4.43 and 5.16 µM, respectively. Conclusion: The results suggested that S₂ subsite is the more selective due to preference by apolar substrates.