Improved Production of Proteases by Brevibacterium linens in Submerged Culture

Abstract

Protease formation in submerged cultivations of Brevibacterium linens was studied The experiments were performed in a highly instrumented fermenter using a mineral salt medium containing 0.5% corn steep liquor (CSL) as sole carbon and nitrogen source. After 20 h of cultivation at 30 °C under an aeration rate of 0.33 vvm the organism produced a proteolytic activity of 33.1 U ml⁻¹. By continuously monitoring various cultivation parameters (dissolved oxygen, carbon dioxide and oxygen in effluent gas, and amount of acid added) and by regularly retrieving samples from the broth for off-line analysis of ammonia, a-amino nitrogen, amino acids and protease activity, a very clear picture of culture development could be obtained: respiration activity and substrate concentration indicated that the proteases were synthesized during linear growth and attained the maximum activity when entering the stationary phase. As amino acids were almost exclusively used for growth, mineral acid was added to maintain a constant pH, and consequently a considerable increase in the ammonium concentration was observed. With the exception of tyrosine and histidine, all amino acids were completely dissimilated Further shake flask cultivation experiments proved that proteolytic activity can be increased up to 70 U ml⁻¹ by supplying 15 g l⁻¹ CSL.