EFFECT OF CULTURE TEMPERATURE AND DISSOLVED-OXYGEN CONCENTRATION ON EXPRESSION OF ALPHA-AMYLASE GENE IN BATCH CULTURE OF SPORE-FORMING HOST, BACILLUS-SUBTILIS 1A289

Spore-forming recombinant Bacillus subtilis 1A289 containing plasmid p C194 carrying an alpha-amylase encoding gene was used for the producti on of the cloned gene product in batch culture. In flask culture, the alpha-amylase activity at a culture temperature of 45 degrees C, was 6 43 U/ml which was 2-fold higher than that at 37 degrees C. No spore fo rmation was detected in the culture. When the cells were cultured in a jar fermenter at 45 degrees C with a dissolved oxygen (DO) level of 9 0%, the enzyme activity was only 184 U/ml which was only 30% of that i n the flask culture. However, when the DO level was controlled at 5%, the activity was 846 U/ml. In the case of cultures at 45 degrees C wit h a DO level higher than 50%, spore concentration and spore fraction w ere more than 3.3 x 10(8) spores/ml and 85-100%, respectively. These v alues were 10 times higher with respect to spore concentration and 6 t imes higher with respect to the fraction of cells forming spores than those at a DO level of 5%. There were three types of cells in the ferm enter: cells resistant to chloramphenicol and producing alpha-amylase; cells having lost plasmid pC194; cells forming spores. The specific e nzyme activity of vegetative cells was 1.9 kU/mg dry cell weight, whil e that of spores was 0.02 kU/mg dry cell weight. The alpha-amylase gen e expression of the spore-forming recombinant B. subtilis was affected strongly by culture temperature, DO level, and fraction of cells form ing spores.