Enhancement and regulation of extracellular protein production by Bacillus brevis 47 through manipulation of cell culture conditions

Bacillus brevis 47 was cultivated in 2 liter fermentors in semidefined media containing polypeptone with or without glucose or fructose. Neither sugar was essential for growth or extracellular (S-layer) protein production, and 2.5 to 3.0 g/L protein was accumulated in the medium. When present, glucose was used very slowly, however, fructose was used much more quickly. Dramatic changes in metabolic indicators (dissolved oxygen and pH) were seen when fructose became depleted, and protease was produced, decreasing the amount of protein ultimatelv accumulated in the medium. Using the change in dissolved oxygen as a marker for the time of addition, polypeptone, fructose, or both were used to stimulate protein production. With the addition of polypeptone, on stimulation was achieved, but protease production was suppressed. Addition of fructose did result in a small stimulation of protein production (to 5 g/L) if added once. Further additions resulted in more growth, but no increase in protein production. Various combinations of polypeptone and fructose were also used, with the most effective combination (fructose added early, fructose and polypeptone added later) resulting in an accumulation of 15 g/L protein in the medium. This is comparable to that seen when B. brevis 47 is grown in a complex glucose medium and stimulated with polypeptone addition at 21 hours. These results are discussed with respect to the structure and function of S-layer proteins, as well as the use of this organism for the production of heterologous proteins.