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)2HPO4, 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 Csoybeano=20 kg m−3 and initial glucose concentration CGo=8 kg m−3; whereas, addition of the inorganic nitrogen source (NH4)2HPO4 decreased SAP production considerably. Further increase in SAP production (3850 U cm−3) was obtained when sucrose was replaced with glucose at Csucroseo=15 kg m−3 and Csoybeano=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 Csoybeano=15 kg m−3 as 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): Csoybeano=20, Csucroseo=15, CNa2HPO4o=0.021, and CNaH2PO4o=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, Damköhler 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.