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Conversion of subtilisin E to thiolsubtilisin by site-directed mutagenesis Chen, Lu


This thesis presents a procedure of site specific mutagenesis of subtilisin E for reducing its proteolytic activity while retaining its ligase activity in peptide synthesis. A host-vector system was chosen to conduct the DNA manipulation and the gene expression. Subtilisin B gene (aprE) cloned on the plasmid DNA pAP65 was subcloned to a phagemid DNA pUC1 18 to facilitate KunkePs oligonucleotide directed mutagenesis. A new plasmid DNA pAP65D was developed from pAP65 and used to harbor subtilisin E gene and thiolsubtilisin gene. As a host cell, the B. subtilis DB428 strain, which is deficient in three major extracellular protease genes, was transformed with the plasmid DNA for expressing subtilisin E gene and thiolsubtilisin gene. By using this procedure, one of the catalytic active site residues on subtilisin E, serine 221, was replaced by a cysteine residue; thus a mutant product, thiolsubtilisin, was obtained. DNA sequencing confirmed both the subtilisin E gene on pAP65D8 and thiolsubtilisin gene on pAP65D8M. An improved transformation method with higher transformation efficiency was derived. The proteolytic activities of subtilisin E and thiolsubtilisin were compared. The B. subtilis transformants with pAP65D8 containing aprE and pAP65D8M containing thiolsubtilisin gene, developed halos around the colonies on skim milk agar plates. However, the halo produced by colonies with thiolsubtilisin gene was smaller than those with aprE. Subtiisin E and thiolsubtilisin secreted into the culture media were then subjected to a proteolytic activity assay. The proteolytic activity measurement, by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, showed that the proteolytic activity of thiolsubtilisin was about 30% of that of subtiisin E, when culture supernatants were assayed. The ligase activity of subtilisin E and thiolsubtilisin was initially planned to be measured by using a solid phase detection method. However, the solid phase detection method failed in this case due to the fact that the enzymes react with the antibody used in the method. As a preliminary test, the ligase activities of subtilisin E and subtilisin Carlsberg were investigated. It was found that both enzymes can catalyze the formation of the dipeptide Z-L-Phe-Met-O-Me. HPLC was found to be able to detect such formation. Further study on the ligase activity of subtilisin B and thiolsubtilisin is suggested.

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