Heterologous Expression and Enzymatic Characterization of D- Psicose 3-Epimerase

Abstract:

The DPE gene from Clostridium bolteae ATCC BAA-613 was cloned and expressed in Bacillus subtilis WB800. We inserted a P43 promoter directly upstream the DPE geneby virtue of overlap extension PCR，which forming P43-Cb-dpe，and then ligated it into pMA5 shuttle vector thus constructing the pMA5-P43-Cb-dpe expression vector. The recombinant plasmid was imported into Bacillus subtilis WB800 for expression. Compared with single promoter expression system，pMA5-P43-Cb-dpe has a palpable promotion of expression level. The expressed recombinant enzyme was purified andits enzymatic properties were studied. The optimum temperature and pH for recombinant enzyme were 55 ℃ and pH 7.0，respectively. It was highly stable at 30～40 ℃ and pH 6.5～7.5. Co2+ and Mn2+ can significantly enhance enzyme activity. The recombinant enzyme's affinity for D-psicose was high than D-frutcose. Meanwhile，in terms of the kinetic parameters，the catalytic efficiency Kcat/Km is greater when use D-psicose as substrate.