Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD31(5) harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35 degrees C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%-92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12-h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.