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

Abstract

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 (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 was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic protease TlAPA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation. IMPORTANCEThe novel aspartic protease TlAPA1 was identified from T. leycettanus and expressed as a zymogen (proTlAPA1) in P. pastoris. Enzymatic characteristics of the

Highly-Expressed Aspartic Protease from Aspergillus Fumigatus-Mediated Enzymolysis of Silkworm (Bombyx Mori) Pupae Protein

“…4B) after incubation for 1 h at 37°C. The optimum pH from this study is similar to that of aspartic protease produced from, Trichoderma asperellum; pH 4.0 [6], Aspergillus niger; pH 4.0 [42], and higher than protease from T. Leycettanus; pH 3.5 [43], T. leycettanus JCM12802; pH 3.0 [3]. Moreover, at pH 4.0-5.0 it attained maximum stability with over 80% of its initial activity (Fig.…”

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides

“…They have two highly conserved aspartic acid residues located at the center of the active site responsible for their catalytic activity (Guo et al 2020 ). Pepsin-like and chymosin-like aspartic proteases are the two main types of aspartic proteases.…”

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation

“…P. pastoris is widely used as an heterologous expression host because of its endotoxin-free use, high exogenous-protein expression, easy product purification, low production cost, and suitability for high density fermentation [22,23]. There are many examples of aspartic proteases successfully expressed in this host, including Bsapa from Bisporomyces MEY-1 [24], RmproA from Talaromyces leycettanus [25], and MCAP from Mucor circinelloides DSM 2183 [26]. But there are few research on protease hydrolysis of soy protein, and it is an inevitable problem that the DH of soy protein is relative low (generally 5-8%) [27][28][29].…”