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

• Published in: Enzyme and microbial technology 2003-12, Vol.33 (7), p.975-986
• Main Authors: Çalık, Pınar, Çelik, Eda, Telli, İlkin E, Oktar, Ceren, Özdemir, Elif
• Format: Article
• Language: English
• Subjects: Bacillus; Bacteriology; Biological and medical sciences; Casein; Complex medium; Fermentation; Fundamental and applied biological sciences. Psychology; Genetics; Medium; Medium design; Microbiology; Molasses; Oxygen transfer; Protein; Serine alkaline protease; Soybean
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2003.07.002

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. (NH 4) 2HPO 4, 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 (NH 4) 2HPO 4 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 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 Na 2HPO 4 o=0.021, and C NaH 2PO 4 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, 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.