Heterologous Expression,Fermentation Optimization Of Endo S And Its Preliminary Immobilization Study

Endo S is an endoglycosidase derived from Streptococcus pyogenes that specifically hydrolyzes N-linked glycans of immunoglobulin G Fc fragment.Since glycosylation of antibodies has a great impact on antibody-dependent cell-mediated cytotoxicity?ADCC?and complement dependent cytotoxicity?CDC?,it has been developed as a tool for homogeneous glycosylation of antibodies and potential therapeutic drugs for autoimmune diseases,which result in remarkably increasing demand for Endo S.However,the studies about the expression optimization of Endo S and its efficient utilization are not sufficient.In this thesis,a heterologous expression strain of Endo S is constructed,and the fermentation of Endo S is optimized at shake flask level.In the meantime,a natural substrate of Endo S,sialyglycopeptide?SGP?is isolated for the studies of Endo S.Finally,Endo S was immobilized on agarose resin in our preliminary immobilization studies.The main results in this thesis are as follow:SGP with 95%purity was isolated from egg yolk after four steps:organic reagent degreasing,solid phase extraction,semi-preparative high-performance liquid chromatography separation and gel purification.During the process,petroleum ether is selected to replace diethyl ethers which is not safe and with high irritating in previous studies.And the purity of SGP is finally improved after G25 gel purification.The gene of Endo S is inserted into plasmid pET28a and expressed in E.coli BL21?DE3?.Single-factor fermentation medium components and conditions optimization of Endo S is performed at shake flask level.The optimized medium was as following(g·L^-1):beef extract25.4,yeast powder 10.0,NaCl 5.0,glycerol 4.0,pH 8.0.Protein expression is induced by 0.25mM IPTG at 20°C for 24 h with a final level of 225 mg·L^-11 fermentation broth,which increases3.5-fold compared to that before optimization and it is 5.6 times higher than the highest yield reported.Finally,the affinity immobilization of Endo S is achieved by histidine tag on agarose resin.The results demonstrated that the reusability and storage stability perform well,with no significant reduction of enzyme activity after six-run reactions of SGP hydrolysis,which all maintain the yield of more than 90%.Meanwhile,after three months of storage,the hydrolysis yield of SGP can still reach 84%.Moreover,0.21 mg of immobilized Endo S can completely hydrolyze the glycan from 5 mg rituximab antibody at 25 ^oC within 30 min,which indicates that immobilized Endo S retained good hydrolysis ability of glycan from antibodies via this method.The flow micro-reactor is built up for SGP hydrolysis using immobilized Endo S,which can provide rapid separation of carrier from reaction mixture.The results show that this flow micro-reactor can achieve similar hydrolysis yield as batch reaction,which indicates that this facility could be further used for scale-up application of immobilized Endo S in antibodies preparation.