Expression, purification, and characterization of soluble and active glutamate-specific endopeptidase in Bacillus licheniformis and Pichia pastoris

Abstract Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is a serine protease with strong specificity toward Glu residue. The relatively complicated purification procedure for producing soluble active GSE-BL limits its wide application in biocatalysis and organic synthesis. In this study, recombinant gse-bl gene with full-length sequence was firstly expressed in the bacterium B. licheniformis (GSE-BL-B) and the yeast Pichia pastoris (GSE-BL-P) in a soluble and enzymatically active form. Pure GSE-BL-B and GSE-BL-P were obtained with yields of 62.5 mg/L and 78.4 mg/L, respectively, after one-step His 6 tag chromatography. Mature GSE-BL-B and GSE-BL-P, with similar enzymatic properties as matured GSE-BL expressed in Escherichia coli (GSE-BL-E), were obtained with yields of 50.8 mg/L and 63.2 mg/L after different durations of trypsin treatment. PNGase treatment and the corresponding enzymatic assay were conducted to validate the glycolysation of GSE-BL-P. The optimal reaction pH and temperature, as well as the kinetic analysis of GSE-BL-B and GSE-BL-P were investigated. The GSE-BLs expressed in B . licheniformis and P. pastoris are potential alternative for the production of enzymatically active GSE-BL with the advantages of much simpler and more cost-effective procedure. Thus, GSE-BLs can be widely applied in peptide synthesis, peptide recovery and sequence analysis.