Process Biochemistry, Vol.80, 52-57, 2019
Primary structure and characterization of a protease from Bacillus amyloliquefaciens isolated from meju, a traditional Korean soybean fermentation starter
Bacillus amyloliquefaciens FSE-68 isolated from a Korean soybean fermentation starter (meju) produces a distinct alkaline protease, alkaline serine-protease (APR68). The primary structure of APR68 was Determined using a combination of tryptic peptide mapping by LC/ESI-MS/MS analysis and DNA sequencing. The APR68 gene is closely (96.2% identical) related to the gene encoding the B. amyloliquefaciens alkaline protease (also known as subtilisin BPN'). The APR68 gene encodes a 30-amino acid signal peptide, a 77-amino acid propeptide, and a 275-amino acid mature protein with a molecular weight of 27,447 Da. The protein sequence of APR68 differs from that of subtilisin BPN' by six amino acids (Phe-21, Val-79, Gly-161, Asn-181, Ser-194 and Ala-260). Among the six amino acids, Val-79 is near a calcium-binding pocket of APR68 and alters its calcium-binding properties. The protease activity of APR68 was more stable than that of subtilisin BPN' in 2 mM EDTA, and its activity increased by 10.1% in 10 mM CaCl2. These results demonstrate the effect of specific amino acid replacements on calcium ion affinity. The APR68 is a good candidate for application under chelating conditions.