Fed-batch culture with controlled L-amino acid composition was performed to improve production of a recombinant gene product in Bacillus brevis. The maximum recombinant protein (alpha-amylase) level and specific activity increased from 5.14 kU/mL and 0.77 kU/mg dry cell in conventional fed-batch culture to 12.01 kU/mL and 2.64 kU/mg dry cell, respectively, when L-amino acid concentration was controlled at 5 mM using an asparagine (Asn)- and isoleucine (Ile)-enriched nitrogen source. The L-amino acid concentration in the culture was monitored by an automatic biotech analyzer and controlled at 2-20 mM using a mixture of polypeptone and yeast extract. Although L-amino acid concentrations were controlled at low levels, the alpha-amylase activity increased only 1.3 times compared to an uncontrolled batch culture; accumulation of ammonium ion was not reduced. When L-amino acid was controlled at the high level, more cell mass and less recombinant gene product were produced than in those with low control level. To overcome ammonium ion inhibition, the specific amino acids Asn and lie were substituted to improve the production of gene product. Addition of these amino acids to a flask culture led to an improvement in the enzyme production level and specific activity to 2.9 and 5.1 times, respectively, as high as that without them. Both the control of amino acids at low concentrations and the enrichment of Asn and lie were effective for the improvement of recombinant protein production from recombinant B. brevis cells.