5273

4943

كارشناسي ارشد

بيوتكنولوژي كشاورزي

اصفهان: دانشگاه صنعتي اصفهان، دانشكده كشاورزي

1388

هفت،119ص.:مصور،جدول،نمودار

ص.ع.به فارسي و انگليسي

آقافخرميرلوحي،غلامرضا احمديان

كامبيز اكبري

21/3/89

مسعود بهار،حميدرضا رحماني

مهندسي كشاورزي

ID4943

به فارسي و انگليسي: قابل رويت در نسخه ديجيتالي

Surface Display of Chitinase on Bacillus Subtilis using Sortase Enzymatic System in order to produce a kind of Biofungicide Hamid reza fasehee Hr fasihi@yahoo com Date of submission March Department of Plant breeding Isfahan University of Technology Isfahan Iran Degree M Sc Language FarsiA F Mirlohi Ph D Professor Supervisor mirlohi@cc iut ac irG R Ahmadian Ph D Assis Prof Supervisor Gholamreza ahmadian@yahoo comAbstract Nowadays modification of the living cell surface has gained a lot of interest inbiotechnological applications immunology and applied microbiology Because of the greatimportance of bacteria in biotechnological research considerable researches have beencarried out into the field of changing their cell surface Expression of recombinant proteinson 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 covalentanchoring of substrate proteins on their cell wall They recognize an amino acid sequencedesignated 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 peptidecrossbridge linking the peptidoglycan strands in a transpeptidation reaction Bacillussubtilis has been reported to code for two different sortases but the sorting sequencesrecognized by them are yet unknown In this study in order to understand the mechanism ofsortase enzymatic system in B subtilis two experiments were carried out for in vivo and invitro sortase assay For in vivo study a cell wall anchoring vector was made by insertingtwo genes a putative sortase gene yhcS and the coding region of the terminal part of theyhcR gene of B subtilis coding for a putative sortase substrate YhcR that was anchored tothe 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 cellsurface of B subtilis was analyzed using SDS PAGE Western blotting DNS and flowcytometery methods Results showed that chitinase was attached to the cell wall and hadenzymatic activity In the next step of experiment a sortase substrate was desingned thatpossessed a sorting motif in its unstructural domain This substrate and sortase YhcS waspurified using affinity chromatoghraphy In vitro sortase assay was done using sortase YhcSand its substrate The results showed that the sortase YhcS is not functional In vitro andmay require some other compounds for its activity In general the results of this researchelucitated to some extent the sortase enzymatic system in B subtilis in which YhcS andYhcR paly a key role Keywords Surface Display Chitinase Sortase enzymatic system Bacillus subtilis

آقافخرميرلوحي،غلامرضا احمديان

كامبيز اكبري

مسعود بهار،حميدرضا رحماني