Protein-based complex medium design for recombinant serine alkaline protease production

This work reports on the design of a complex medium based on simple and complex carbon sources, i.e. glucose, sucrose, molasses, and defatted-soybean, and simple and complex nitrogen sources, i.e. (NH4)(2)HPO4, casein, and defatted-soybean, for serine alkaline protease (SAP) production by recombinant Bacillus subtilis carrying pHV1431::subC gene. SAP activity was obtained as 3050 U cm(-3) with the initial defatted-soybean concentration C-soybean(o) = 20 kg m(-3) and initial glucose concentration C-G(o) = 8 kg m(-3); whereas, addition of the inorganic nitrogen source (NH4)(2)HPO4 decreased SAP production considerably. Further increase in SAP production (3850 U cm(-3)) was obtained when sucrose was replaced with glucose at C-sucrose(o) = 15 kg m(-3) and C-soybean(o) = 20 kg m(-3). Nevertheless, when molasses was replaced with sucrose, the maximum activity was obtained with molasses having 10 kg m(-3) initial sucrose concentration and C-soybean(o) = 15 kg m(-3) as soybean 2130 U cm(-3); moreover, when casein was replaced with defatted-soybean SAP production decreased considerably (ca. 250 U cm(-3)). Thereafter. the effects of inorganic ionic compounds were investigated; and except phosphate, inorganic compounds supplied from defatted-soybean were found to be sufficient for the bioprocess. The highest SAP activity was obtained as 5350 U cm(-3) in the medium that contained (kg m(-3)): C-soybean(o) = 20, C-sucrose(o) = 15, C-Na2HPO4(o) = 0.021, and C-NaH2PO4(o) = 2.82 that was 6.5-fold higher than that of the SAP produced in the defined medium. By using the designed complex medium, oxygen transfer characteristics of the bioprocess were investigated; and, Damkohler number that is the oxygen transfer limitation increases with the cultivation time until t = 14 h; and, at t > 20 h both mass transfer and biochemical reaction resistances were effective. Overall oxygen transfer coefficient varied between 0.010 and 0.044 s(-1); volumetric oxygen uptake rate varied between 0.001 and 0.006 mol m(-3) s(-1); and specific oxygen uptake rate varied between 0.0001 and 0.0022 mol kg(-1) DW s(-1) throughout the bioprocess. (C) 2003 Elsevier Inc. All rights reserved.