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

• Published in: Enzyme and microbial technology Vol. 33; no. 7; pp. 975 - 986
• Main Authors: Çalık, Pınar, Çelik, Eda, Telli, İlkin E, Oktar, Ceren, Özdemir, Elif
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 02-12-2003; Elsevier Science
• 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; Molasses; Casein; Medium design; Serine alkaline protease; Medium; Bacillus; Oxygen transfer; Soybean; Fermentation; Complex medium; Protein; Bacillaceae; Bacillales; Dicotyledones; Angiospermae; Production; Bacteria; Recombinant protein; Culture medium; Serine endopeptidases; Enzyme; Glycine max; Peptidases; Leguminosae; Hydrolases; Spermatophyta
• 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.