Cloning,Expression,and Characterization of Serine Protease AprX from Geobacillus thermoleovorans ARTRW1

Abstract

A novel thermostable protease, AprX from G. thermoleovorans ARTRW1, was recombinantly produced, purified and characterized. This work shows that the 85 amino acids from the N-terminal was cleaved post-translationally, indicating that the enzyme was synthesized as an inactive precursor “zymogen”. The molecular weight of the mature protein is 38.1 kDA. Prolonged incubation at different temperatures and time intervals showed that the protease caused self-cleavage above 45°C and AprX degraded completely within 6 h. Mass spectrometry analysis has shown that the enzyme has a partial preference to cleave after R, K and L residues similar to trypsin but it also cleaves after the C-terminal end of E, S, V, F, G, H, N and T residues. The enzyme activity reached maximum at 55°C and over a broad pH range between 5 and 11. The protease was found to be highly tolerant of detergents and completely inhibited by PMSF, Zn²⁺ and Ni²⁺, similar to trypsin-like serine proteases. It was stable from 30°C to 70°C, retaining 80% activity for 3 h at 55°C. This new protease could be a candidate for use in a variety of industrial processes that require long-term stability at elevated temperatures.