Construction and Application of a Multienzyme System for Synthesis of L-malate.

This study aimed to develop a multienzymatic system for synthesis of L-malate. First, recombinant Escherichia coli strains were constructed expressing maleic acid cis-trans isomerase (MaiA) or fumarase C (FumC) from different sources. Serratia marcescens MaiA (SMaiA) and E. coli FumC (ECFumC) showed good catalytic performance. Next, six co-expression systems for SMaiA and ECFumC were constructed. E. coli BL21 (DE3)-pRSFDuet-1-ecfumC-smaiA (named strain pFM2) had the highest L-malate catalytic activity. In 7-L fed-batch fermentation, the SMaiA and ECFumC activities of strain pFM2 wet cells were 43.4 and 154.5 U/g, respectively, 2.4- and 10.7-fold the values that were obtained in shaken flasks. Finally, a whole-cell catalytic process was established for the production of L-malate by strain pFM2 with maleate as the substrate. When the dose of pFM2 wet cells was 0.5 g/100 mL and 1 mol/L maleate was the substrate, the catalytic process was completed within 4 h. Notably, the intermediate fumarate was almost absent during the conversion process. The concentration of L-malate reached 143.8 g/L with a yield of 0.60 g/(L·min). The molar conversion rate of the substrate was 98.4%. These findings lay a foundation for the industrial application of multienzymatic synthesis of L-malate.

Biochemical Characterization and Synthetic Application of WciN and Its Mutants From Streptococcus pneumoniae Serotype 6B.

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.

Immunogenicity Assessment of Different Segments and Domains of Group a Streptococcal C5a Peptidase and Their Application Potential as Carrier Protein for Glycoconjugate Vaccine Development.

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.