The biochemical property and functional identification of three recombinant glycosyltransferases, including β-1,4-rhamnosyltransferase (Cps8R), β-1,4-galactosyltransferase (Cps8J) and α-2,3-sialyltransferase (Cps8K) involved in the biosynthesis of the tetrasaccharide repeating unit of serotype VIII capsular polysaccharide (CPS) of Group B Streptococci (GBS), were systematically investigated. Subsequently, these recombinant enzymes were employed for one-pot three-enzyme efficient synthesis of the tetrasaccharide repeating unit of GBS serotype VIII CPS using the chemically synthesized Glcα-PP-(CH 2 ) 11 -OPh as the starting substrate in a satisfying yield of 87%.

The enzymatic activity and function of a new α-1,3-glucosyltransferase, Cps18CU, derived from Streptococcus pneumoniae serotype 18C was identified for the first time using the enzymatically synthetic disaccharide Rhaβ1,4-Glcα-PP-O(CH 2 ) 11 -OPh as the acceptor substrate. Further investigation on substrate specificity revealed that Cps18CU exhibited broad toleration toward various NDP-Glc donors and also accepted such disaccharide acceptor containing Rhaβ1,4-Glc moiety in structure. Finally, Cps18CU was employed into a one-pot twoenzyme reaction system, furnishing trisaccharide Glcα1,3-Rhaβ1,4-Glcα-PP-O(CH 2 ) 11 -OPh in an 81 % yield.

Group A streptococcal C5a peptidase (ScpA) is a highly conserved surface virulence factor present on group A streptococcus (GAS) cell surfaces. It has attracted much more attention as a promising antigenic target for GAS vaccine development due to its high antigenicity to stimulate specific and immunoprotective antibodies. In this study, a series of segments of ScpA were rationally designed according to the functional domains described in its crystal structure, efficiently prepared and immunologically evaluated so as to assess their potential as antigens for the development of subunit vaccines. Immunological studies revealed that Fn, Fn2, and rsScpA193 proteins were promising antigen candidates worthy for further exploration. In addition, the potential of Fn and Fn2 as carrier proteins to formulate effective glycoconjugate vaccine was also investigated.

The biochemical properties of α-1,3-galactosyltransferase WciN from Streptococcus pneumoniae serotype 6B were systemically characterized with the chemically synthesized Glcα-PP-(CH2)11-OPh as an acceptor substrate. The in vitro site-directed mutation of D38 and A150 residues of WciN was further investigated, and the enzymatic activities of those WciN mutants revealed that A150 residue was the pivotal residue responsible for nucleotide donor recognition and the single-site mutation could completely cause pneumococcus serotype switch. Using WciNA150P and WciNA150D mutants as useful tool enzymes, the disaccharides Galα1,3Glcα-PP-(CH2)11-OPh and Glcα1,3Glcα-PP-(CH2)11-OPh were successfully prepared in multi-milligram scale in high yields.