Cloning And Expression Of Mandelate Racemase Gene And S-mandelate Dehydrogenase Gene

Phenyl gyoxilic acid (PGA), also called benzoylformic acid (BFA) is applied widely as an organic synthetic intermediate. It can be used to synthesize a lot of drugs and pesticide. Complexation with metal elements, it can also be used as sensitizer of fluorescence materials and catalyst of organic oxidation reaction. As a result of that, it attracts more and more attention now.Mandelate racemase (MR) and mandelate dehydrogenase (MDH) are two critical enzymes for mandelic acid to produce PGA in microbial metabolism. According to the results of previous studies, when mandelic acid was used as the only carbon source, strain of high mandelic acid transformation activity can be obtained. The strain was proved as Pseudomonas aeruginosa according to the identification of the No.62 strain, one of the active strains among it. The experiment shows that the No. 62 strain can convert R-mandelic acid and S-mandelic acid simultaneously. It also proves that the No.62 strain is an ideal strain to clone mandelate racemase gene (mdlA) and mandelate dehydrogenase gene (mdlB).The gene of mdlA and mdlB was amplified from No.62 strain by PCR and was cloned into vector pMD18-T to construct the recombinant plasmid pT-mdlA+mdlB; then, it was subcloned into NdeI/NotI sites of pET30a (+) to construct the recombinant plasmid pET30a-mdlA+mdlB. The recombinant strain was induced by 0.2mM IPTG, SDS-PAGE showed that the molecular weight of the proteins expressed was about 43KDa and 38KDa, which was accordant with the theoretical value.Primary studies on recombinant strain whole-cell convert mandelic acid to produce PGA shows that when 0.2mM IPTG (or 0.5% lactose) was used as inducer and the substrate concentration was 1%, the conversion ration of recombined strain can be 97.40% in 48h. The efficiency can be higher when free cells were used and it is greatly influenced by the substrate concentration.