Expression Of Hydroxymandelate Synthase And Mandelic Acid Biosynthesis

Hydroxymandelate synthase (HmaS) is an important dioxygenase that catalysesphenylpyruvate to synthesize mandelic acid. HmaS has a unique function with substratedecarbonization, which determines its important application value in the microbial metabolicpathway and organic synthesis. Therefore, it becomes one of research hottest topics inenzyme engineering in recent years. In this research,based on the aromatic amino acidsbiosynthesis pathway of recombinant strain E. coli W3110/pR15ABK which can producephenylalanine, the synthetic pathway of mandelic acid was constructed by the introduction ofexogenous hydroxymandelate synthase gene. The main results were as follows:(1)The coding sequences of hmas genes from Amycolatopsis orientalis and Streptomycescoelicolor were respectively amplified by the genomic DNAs and were heterologousexpressed. Hmas was expressed in Escherichia coli by means of enzyme activity detectionand identification of SDS-PAGE.(2)Crude enzyme solution from different recombinants were isolated and purified bymethods of anion exchange chromatography (HiPrep16/10DEAE FF) and Gel filtrationchromatography (Sephadex G-75). Then the enzymatic activity of HmaS were evaluated. Theresults showed as follows:①HmaSAO: the specific activity153.91U/mg, purification flod12.79, the recovery32.43%;②HmaSSC1: the specific activity505.31U/mg, purification flod22.61, the recovery27.09%;③HmaSSC2: pecific activity557.25U/mg, purification flod20.91, the recovery26.48%.(3)According to the analysis of characterization of recombinant hydroxymandelatesynthase, the optimum reaction temperature of HmaSAOis28°C. The enzyme activity canalso be more than50%under45°C, and it maintains highest activity at pH8.0with a wellfunction under alkaline conditions. HmaSSC1has the optimum reaction temperature of30°C,more than50%residual activity under60°C with some extends heat resistance. And itmaintains highest activity at pH7.5with well function under slightly alkaline conditions.While the optimum reaction temperature of HmaSSC2is35°C, The residual enzyme activitystill can reach more than50%under65°C.The heat resistance of HmaSSC2is better than theHmaSSC1, with the highest enzyme activity at pH7.0, which performs a specific functionunder neutrality conditions. HmaSSC2is more suitable for altering Escherichia coli bymetabolic engineering to produce mandelic acid.(4) Recombination strain E. coli W3110/pR15ABKHmaS2was reconstructed by linkingthe hmaSSC2gene to the plasmid of pR15ABK. Results of shake-flask fermentation test:biomass logarithmic phase10~48h, the stable phase about48h and the cell concentration(OD600)7.78, mandelic acid41mg/L after60h. The results of15L bioreactor fermentation test: biomass logarithmic phase12~43h, stable phase43h and after52h, the cellconcentration (OD600)101.25, mandelic acid748mg/L.