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Jason M. Herr

   Graduate Research Assistant

    Dept of Chemistry and Chemical Engineering

    South Dakota School of Mines and Technology

    501 East St.Joseph Street 

    Rapid City, SD 57701

    Phone: 605-431-9669

    Fax   :605-394-1232

    Email: liljasonherr@hotmail.com

High Cell Density Fermentations To Optimize Monooxygenase Enzyme Production From Recombinant E.coli

The meta isomer of hydroxydiphenylacetylene (HDPA) is a vital precursor in the production of thermally stable polymers withstanding temperatures as high as 265 °C.Biocatalysis experiments using E. coli TOP10F’with the recombinant plasmid pJS1409 containing a DNA fragment encoding for toluene/benzene-4-monooxygenase (Tb4m) have yielded 75 % meta-HDPA purities. However, reaction rates are low at 0.14 μmol/L/min due to low substrate solubility and product inhibition at levels as low as 10 μmol/L.The goal of this research is to maximize the monooxygenase enzyme levels as a means of enhancing m-HDPA biocatalysis rates.  High cell density fed-batch systems increase biocatalysis rates by increasing the cell density and, in turn, the total enzyme concentration.  Three strategies were investigated in this research: medium optimization, fed-batch optimization (specific growth rate vs. max cell density), and time of induction.  An optimized base and feed medium composition was determined using factorial design methods.  Fed-batch systems were then run at various specific growth rates to maximize the final cell density for the fermentation.  Finally, the effect of induction time on specific and volumetric monooxygenase activity was investigated.