A novel thermostable aspartic protease from Talaromyces leycettanus and its specific autocatalytic activation through an intermediate transition state.

Aspartic proteases exhibit optimum enzyme activity under acidic conditions and have been extensively used in food, fermentation, and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) from Talaromyces leycettanus was identified and successfully expressed in Pichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions. TlAPA1 shared the highest identity of 70.3% with the aspartic endopeptidase from Byssochlamys spectabilis (GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. Mature TlAPA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature of TlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45-kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates and activation of the mature protein (TlAPA1). The former step can be processed without proteolytic activity, while the latter process depended on protease activity completely. The discovery of the novel aspartic protease TlAPA1 and the study of its activation process will contribute to a better understanding of the mechanism of aspartic protease auto-activation.