Enhanced amylase production by Bacillus subtilis using a dual exponential feeding strategy.

A recombinant Bacillus subtilis strain (ATCC 31784) haboring the plasmid pC194 with a thermostable alpha-amylase gene was cultured in a 22-l B. Braun Biostat C fermenter. Traditional batch operations suffer from low cell mass and protein productions because a high initial glucose concentration causes substrate inhibition and also product inhibition due to acetate accumulation. An exponential fed-batch strategy to prevent these inhibitions was developed in this work. The host strain is auxotrophic for phenylalanine, tyrosine and tryptophan. Due to low solubilities of tyrosine and tryptophan in the feed stream, tyrosine and tryptophan were dissolved separately in ammonia water to form a second feed stream. By dual feeding both streams at different exponential feed rates, a high cell density of 17.6 g/l and a final alpha-amylase activity of 41.4 U/ml and the overall biomass yield of 0.39 g cell/g glucose were achieved.