Effective extracellular production of Bacillus stearothermophilus esterase by pH-stat modal fed-batch culture of recombinant Bacillus brevis.

An automated two-component substrate feeding strategy with a pH-stat modal fed-batch culture using a high pH limit was developed to effectively produce esterase from a hyperprotein-excreting Bacillus brevis HPD31 harboring a plasmid pHSC131 which carries a Bacillus stearothermophilus esterase gene. First, the effect of single- and multi-substrate feedings on the growth and activity of the excreted esterase was investigated. Then a two-component (polypepton + glucose) feeding using different feed rates was studied. Highest activity of the excreted esterase (34 U/mL) was obtained when the concentrations of polypepton and glucose in the nutrient feed solution were 250 and 41.60 g/L respectively. The absence and excessive amount of glucose in the nutrient feed solution was ineffective for the extracellular esterase formation because without glucose the increase in cell concentration was minimum while excessive amount of glucose favored cell growth at the expense of the esterase production. It is believed that the mechanism of enzyme excretion is growth dependent and that a higher cell growth of the host is in effect unfavorable for the enzyme production. The feed rate, automatically controlled by the direct signal of the pH change, at 0.30 mL/pulse was found optimum for the esterase production while lower (0.15 mL/pulse) and higher (0.67 mL/pulse) feed rates did not produce good results. The activity of the excreted esterase was increased more than eight times from 4 U/mL obtained in the conventional batch culture to 34 U/mL obtained in this study. The esterase productivity was likewise increased more than threefold. (c) 1992 John Wiley & Sons, Inc.