Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp.

Telli, İlkin Ece
In this study, firstly, bioprocess characteristics for Serine Alkaline Protease (SAP) production, using recombinant Bacillus subtilis carrying pHV1431::subC, were examined. The cell concentration, substrate concentration, SAP activity and SAP synthesis rate profiles demonstrated that the system reaches to a steady state in terms of cell growth and SAP synthesis between t=15-25 h, therefore, this time interval is appropriate to employ both metabolic flux analysis and metabolic control analysis, which apply strictly to steady state systems. After that, three separate perturbations were introduced by addition of aspartate to the production medium at a certain time of the bioprocess. The response of the cells were observed and; by comparing the changes in intracellular reactions of aspartate pathway, Asn, Thr and Ile productions were determined to be the bottlenecks in aspartate pathway and the branchpoints splitting from Asp and AspSa were identified to be weakly rigid branchpoints. Lastly, metabolic control analysis principles were applied to determine the elasticity and flux control coefficients of the simplified aspartate pathway. Aspartate formation reaction and Lys, Thr, Ile, Met producing group share the control of asparagine synthesis. The results revealed that lysine producing branch flux dominates the other branch fluxes, therefore to eliminate bottlenecks and increase SAP production, the activity of the branches leading to the formation of Asn, Thr and Ile should be increased while decreasing the activity of lysine synthesizing branch. This could be achieved either by genetic manipulation or by addition of specific inhibitors or activators to the system.


Comparison of benzaldehyde lyase production capacity in recombinant Escherichia coli and recombinant Bacillus species
Kaya, Hande; Çalık, Pınar; Department of Chemical Engineering (2006)
In this study, the benzaldehyde lyase (BAL, EC production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3)...
Recombinant therapeutic protease production by Bacillus sp.
Korkmaz, Nuriye; Çalık, Pınar; Department of Chemical Engineering (2007)
The first aim of this study is the development of extracellular recombinant therapeutic protease streptokinase producing Bacillus sp., and the second aim is to determine fermentation characteristics for streptokinase production. In this context, the signal (pre-) DNA sequence of B.licheniformis (DSM1969) extracellular serine alkaline protease enzyme gene (subC: Acc. No. X03341) was ligated to 5’ end of the streptokinase gene (skc: Acc. No. S46536) by SOE (Gene Splicing by Overlap Extension) method through P...
Determination and metabolic engineering of rate limiting reactions in aromatic amino acid pathway in Bacillus subtilis for L-phenylaianine production
Guzide, Calik; Yasemin, Demirci; Pinar, Calik; Ozdamar, Tuncer H. (2007-09-01)
Rate limiting reactions in the aromatic-group amino acid pathway (AAAP) in Bacillus subtilis for l-phenylalanine (Phe) production were determined as reported elsewhere (Özçelik-Şenver et al., 2004). The biochemical reactions in the AAAP start with the reaction using phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) synthesised in the glycolysis pathway and the PPP, respectively, leading to the formation of Phe via chorismate by branching at prephenate. The branch-point metabolites E4P supplied in vi...
Growth of agriculturally important Pseudomonas spp. and Azotobacter Chroococcum on beer waste and observation of their survival in peat
Abat, Benek; Hamamcı, Haluk; Department of Food Engineering (2006)
In this study agriculturally important Pseudomonas spp. which may solubilize phosphate and Azotobacter chroococcum which can fix atmospheric nitrogen were grown on waste beer with 4 different concentrations and conditions for best growth were determined. Having potential of use as biofertilizers, they were put in the carrier material peat and survivals of them were observed for 3 months at three different temperatures. Biofertilizer can be defined as a substance which contains living microorganisms which, w...
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...
Citation Formats
İ. E. Telli, “Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp.,” M.S. - Master of Science, Middle East Technical University, 2004.