A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Aspartic proteases exhibit optimum enzyme activity under acidic condition and have been extensively used in food, fermentation and leather industries. In this study, a novel aspartic protease precursor (pro<jats:italic>Tl</jats:italic>APA1) from <jats:italic>Talaromyces leycettanus</jats:italic> was identified and successfully expressed in <jats:italic>Pichia pastoris</jats:italic>. Subsequently, the auto-activation processing of the zymogen pro<jats:italic>Tl</jats:italic>APA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions. <jats:italic>Tl</jats:italic>APA1 shared the highest identity of 70.3 % with the aspartic endopeptidase from <jats:italic>Byssochlamys spectabilis</jats:italic> (GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. Mature <jats:italic>Tl</jats:italic>APA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature of <jats:italic>Tl</jats:italic>APA1 aspartic protease. During the auto-activation processing of pro<jats:italic>Tl</jats:italic>APA1, a 45 kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates, and activation of the mature protein (<jats:italic>Tl</jats:italic>APA1). The former step was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic protease <jats:italic>Tl</jats:italic>APA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation.</jats:p><jats:sec><jats:title>IMPORTANCE</jats:title><jats:p>The novel aspartic protease <jats:italic>Tl</jats:italic>APA1 was identified from <jats:italic>T. leycettanus</jats:italic> and expressed as a zymogen (pro<jats:italic>Tl</jats:italic>APA1) in <jats:italic>P. pastoris</jats:italic>. Enzymatic characteristics of the mature protein were studied and the specific pattern of zymogen conversion was described. The auto-activation processing of pro<jats:italic>Tl</jats:italic>APA1 proceeded in two stages and an intermediate was identified in this process. These results describe a new subgroup of aspartic protease A1 family and provide insights into a novel mode of activation processing in aspartic proteases.</jats:p></jats:sec>