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

Çalık, Pınar
Telli, IE
Oktar, C
Ozdemir, E
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.


Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses
Çalık, Pınar; Ozdamar, TH (Elsevier BV, 2003-12-02)
Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli-Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP product...
Mass flux balance-based model and metabolic pathway engineering analysis for serine alkaline protease synthesis by Bacillus licheniformis
Çalık, Pınar (Elsevier BV, 1999-07-01)
A mass flux balance-based stoichiometric model of Bacillus licheniformis for the serine alkaline protease (SAP) fermentation process has been established. The model considers 147 reaction fluxes, and there are 105 metabolites that are assumed to be in pseudo-steady state. Metabolic flux distributions were obtained from the solution of the model based on the minimum SAP accumulation rate assumption in B. licheniformis in combination with the off-line extracellular analyses of the metabolites that were the so...
Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network
Çalık, Pınar; Oliver, SG; Ozdamar, TH (Elsevier BV, 2003-05-20)
This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes wer...
Targeted disruption of homoserine dehydrogenase gene and its effect on cephamycin C production in Streptomyces clavuligerus
Yilmaz, Ebru I.; Caydasi, Ayse K.; Özcengiz, Gülay (Springer Science and Business Media LLC, 2008-01-01)
The aspartate pathway of Streptomyces clavuligerus is an important primary metabolic pathway which provides substrates for beta-lactam synthesis. In this study, the hom gene which encodes homoserine dehydrogenase was cloned from the cephamycin C producer S. clavuligerus NRRL 3585 and characterized. The fully sequenced open reading frame encodes 433 amino acids with a deduced M (r) of 44.9 kDa. The gene was heterologously expressed in the auxotroph mutant Escherichia coli CGSC 5075 and the recombinant protei...
Oxygen transfer effects in serine alkaline protease fermentation by Bacillus licheniformis: Use of citric acid as the carbon source
Çalık, Pınar (Elsevier BV, 1998-12-01)
The effects of oxygen transfer on serine alkaline protease (SAP) production by Bacillus licheniformis on a defined medium with C-c = 9.0 kg m(-3) citric acid as sole carbon source were investigated in 3.5 dm(3) batch bioreactor systems. The concentrations of the product (SAP) and by-products, i.e., neutral protease, amylase, amino acids, and organic acids were determined in addition to SAP activities. Ar Q(o)/V = 1 vvm airflow rate, the effect of agitation rate on DO concentration, pH, product, and by-produ...
Citation Formats
P. Çalık, I. Telli, C. Oktar, and E. Ozdemir, “Protein-based complex medium design for recombinant serine alkaline protease production,” ENZYME AND MICROBIAL TECHNOLOGY, pp. 975–986, 2003, Accessed: 00, 2020. [Online]. Available: