Nowadays, modification of the living cell surface has gained a lot of interest in biotechnological applications, immunology and applied microbiology. Because of the great importance of bacteria in biotechnological research, considerable researches have been carried out into the field of changing their cell surface. Expression of recombinant proteins on the cell surface is one of the strategies to change the bacterial cell surface. Gram-positive bacteria code for one or more enzymes termed sortases which catalyze the covalent anchoring of substrate proteins on their cell wall. They recognize an amino acid sequence designated sorting motif, present close to the C-terminal end of the substrate proteins, cleave within this motif and catalyze anchoring of the polypeptide chain to the peptide croridge linking the peptidoglycan strands in a traeptidation reaction. Bacillus subtilis has been reported to code for two different sortases but the sorting sequences recognized by them are yet unknown. In this study in order to understand the mechanism of sortase enzymatic system in B. subtilis , two experiments were carried out for in vivo and in vitro sortase assay. For in vivo study a cell wall anchoring vector was made by inserting two genes, a putative sortase gene yhcS and the coding region of the 3‘ terminal part of the yhcR gene of B. subtilis (coding for a putative sortase substrate YhcR) that was anchored to the 3’ end of Chitinase gene (ChiS). Cell wall anchoring vector was transformed to the B. subtilis and the cells were cultured in LB medium. The presence of Chitinase on the cell surface of B. subtilis was analyzed using SDS-PAGE, Western blotting DNS and flow cytometery methods. Results showed that chitinase was attached to the cell wall and had enzymatic activity. In the next step of experiment, a sortase substrate was desingned that possessed a sorting motif in its unstructural domain. This substrate and sortase YhcS was purified using affinity chromatoghraphy. In vitro sortase assay was done using sortase YhcS and its substrate. The results showed that the sortase YhcS is not functional I vitro . Keywords : Surface Display, Chitinase, Sortase Enzymatic system, Bacillus subtilis
