RESUMEN
To improve the specific activity and catalytic efficiency of l-LcLDH1, an NADH-dependent allosteric l-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1Q88R, Lcldh1I229A and Lcldh1T235G, were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, l-LcLDH1Q88R or l-LcLDH1I229A, displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded l-phenyllactic acid (PLA) with an enantiomeric excess (eep) more than 99%. Their catalytic efficiencies (kcat/Km) without d-fructose-1,6-diphosphate (d-FDP) were 4.8- and 5.2-fold that of l-LcLDH1. Additionally, the kcat/Km values of l-LcLDH1Q88R and l-LcLDH1I229A with d-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without d-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in l-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of l-LcLDH1.
Asunto(s)
Proteínas Bacterianas , L-Lactato Deshidrogenasa , Lacticaseibacillus casei/enzimología , Ácidos Fenilpirúvicos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Escherichia coli/genética , Escherichia coli/metabolismo , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Ácidos Fenilpirúvicos/química , Ingeniería de ProteínasRESUMEN
Vaccines are critical tools for the prevention and treatment of several diseases. Adjuvants have been traditionally used to enhance immunity to vaccines and experimental antigens. In the present study, the adjuvant combination of CpG oligodeoxynucleotides (CpG ODN) and the innate defense regulator (IDR) peptide, IDRHH2, was evaluated for its ability to enhance and modulate the immune response when formulated with alum and the recombinant hepatitis B surface antigen (HBsAg). The CpGHH2 complex enhanced the secretions of tumor necrosis factorα, monocyte chemotactic protein 1 and interferonγ by human peripheral blood mononuclear cells and promoted murine bone marrow dentritic cell maturation. In addition, the present study demonstrated that IDRHH2 was chemotactic for human neutrophils, THP1 cells and RAW264.7 cells at concentrations between 2.5 and 40 µg/ml. The present study also observed that signiï¬cantly higher antiHBs antibody titers, which were sustained at high levels for as long as 35 weeks following the boost immunization, were induced by the combination adjuvant, even when coadministered with a commercial hepatitis B vaccine at a low antigen dose (0.1 µg HBsAg). Notably, the level of IgG2a was almost equal to the level of IgG1, indicating that a balanced T helper (Th)1/Th2 immune response was elicited by the novel vaccine, which was consistent with the ELISpot results. These data suggest that the CpGHH2 complex may be a potential effective adjuvant, which facilitates a reduction in the dose of antigen and induces longlasting, balanced immune responses.