Investigation of bioreactor operation conditions for serine alkaline protease production by Bacillus subtilis

Özdemir, Elif
In this study, complex medium was designed for increasing serine alkaline protease (SAP) production by recombinant Bacillus sp. In this context, the effects of glucose, sucrose, inorganic nitrogen source, casein, defatted soybean, and molasses and their concerted effects were investigated in agitation (N=200 min"1) and heating rate (T=37°C) controlled laboratory scale bioreactors. For this purpose, experiments were started by microorganism selection. It was found that, most suitable microorganism for complex medium was r-B.subtilis carrying pHV1431. ;:subC gene. After considering soybean as a nitrogen source, the effects of (NH4)2HP04 concentration were investigated. As maximum SAP activity was obtained in the medium which did not contain (NH4)2HP04, soybean was selected as the sole nitrogen source. Then, binary effects of defatted soybean and glucose; defatted soybean and sucrose; defatted soybean and molasses; and casein and sucrose were investigated. Among these, maximum activity was obtained as 3852 U cm"3 at t=46 h with CSOybean0 =20 kg m"3 and CSUcrose°=15 kg m"3. Thereafter, effects of inorganic compounds were investigated and it was found that except phosphate, inorganic compounds found in soybean source were sufficient for SAP production, and the activity increased to 5347 U cm"3 with the optimization of the phosphate concentration. Finally, by using the medium designed the variations in the cell growth, protease production, oxygen uptake rate, and oxygen transfer coefficient with the cultivation time were determined in 2.0 dm3 bioreactor systems having temperature, pH, foam, air inlet and stirring rate controls. The maximum SAP activity reached a maximum at t=27 h with a value of 4737 U cm"3 and mass transfer coefficient varied between 0.0096 -0.0435 s"1; whereas, volumetric oxygen uptake rate varied between 0.001-0.006 mol m"3 s'1; and specific oxygen uptake rate varied between 0.0001-0.0022 mol kg"1 s1 throughout the bioprocess.


Determination of metabolic bottlenecks using reaction engineering principles in serine alkaline protease production by recombinant bacillus sp.
Telli, İlkin Ece; Çalık, Pınar; Department of Chemical Engineering (2004)
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 s...
Protease secretion capacity and perforce analysis of recombinant Bacillus species
Çalık, Pınar; Ozdamar, TH (2000-10-08)
Recombinant 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. subC gene was amplified from the chromosomal DNA of the wild-type Bacillus licheniformis by using PCR technology; thereafter, subC gene was first cloned into the pRS316 E. coli yeast shuttle plasmid, then sub-cloned into the pHV1431 E. coli-Bacillus shuttle vector, and transferred to the host Bacillus species, i.e. ...
Bioprocess development for serine alkaline protease production: A review
Çalık, Pınar; Ozdamar, TH (2001-01-01)
This work is the result of the convergence of the research in biochemical reaction engineering and metabolic flux analysis for serine alkaline protease (SAP) production. The genus Bacillus includes a variety of industrially important species that are known to secrete a large number of extracellular proteases and are used among many species as producer of SAP enzyme. Therefore, in the first part of the present article an overview to serine alkaline protease and regulation of its synthesis and secretion in Ba...
Influence of pH conditions on metabolic regulations in serine alkaline protease production by Bacillus licheniformis
Çalık, Pınar; Calik, G; Ozdamar, TH (2002-10-03)
The effects of the controlled and uncontrolled pH conditions, as well as of the value of initial pH in the range of 7-7.5, on serine alkaline protease (SAP) production by Bacillus licheniformis were investigated on a defined medium with the single carbon source glucose in batch bioreactors. Besides uncontrolled pH operations, growth phase, production phase, and entire process control strategies were also applied. The concentrations of the product (SAP) and by-products, i.e. neutral protease, amylase, amino ...
Comparative analysis of product and by-product distributions in defined and complex media in serine alkaline protease production by recombinant Basillus subtilis
Oktar, Ceren; Çalık, Pınar; Department of Chemical Engineering (2003)
In this study, firstly the effects of aspartic acid group amino acids -which were reported to be the potential bottleneck in serine alkaline protease (SAP) synthesis- on SAP production were investigated by substituting at a concentration range of 0-15 mM by using recombinant Bacillus subtilis carrying pHV1434::subC gene. All aspartic acid group amino acids except threonine inhibited SAP activity when CAA= 2.5 mM. The highest SAP activities with asparagine, aspartic acid, lysine, threonine, isoleucine and me...
Citation Formats
E. Özdemir, “Investigation of bioreactor operation conditions for serine alkaline protease production by Bacillus subtilis,” M.S. - Master of Science, Middle East Technical University, 2003.