Enhanced production of α‐amylase in fed‐batch cultures of Bacillus subtilis TN106[pAT5]

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Abstract
Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid‐encoded protein (α‐amylase) are investigated in batch, continuous, and fed‐batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of α‐amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of α‐amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed‐batch experiments with constant volumetric feed rate demonstrate that α‐amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable α‐amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed‐batch experiments, production of α‐amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two‐stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and α‐amylase production in the second stage, in attaining increased bulk α‐amylase activity is demonstrated. © 1993 John Wiley & Sons, Inc.