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.

Serine alkaline protease (SAP, EC 3.4.21.62) produced by Bacillus species, that are the microbioreactors within the bioreactors, is one of the major industrial enzymes. In this study, after production by Recombinant Bacillus subtilis (BGSC-1A751), carrying pHV1431::subc gene in the complex media and separation of solids, SAP was dried by using a spray drier. Experiments were performed to investigate the stabilization of SAP during spray drying and subsequent storage. Initially, the effect of air inlet temperature of the spray drier on SAP activity was evaluated. For this purpose, SAP solutions were spray dried in the absence of any protective agents at five different air inlet temperature (700C, 900C, 1100C, 1200C, 1300C). As a result, increasing air inlet temperature lead to an increase in activity loss of SAP during drying. Thereafter, the effect of protective additives, glucose and maltodextrin (0.5%, 1%, 2% w/v), on SAP activity was investigated during spray drying. The activity loss of SAP was completely inhibited in the presence of glucose at 70, 90 and 1100C. However, the addition of maltodextrin was better than glucose for activity preservation of SAP at 1300C. Among the obtained results, 1% glucose addition was the best to preserve activity of SAP during spray drying. Then, structural change of SAP during drying was investigated. FTIR-ATR spectrum was used to evaluate the change in physical structure of the dried SAP powders in the presence of 1% glucose. From infrared images, at 900C more native-like structure for dried SAP powders was observed. In the last stage of this study storage stability of obtained SAP powders at 40C for a long period (6 months) was investigated. When considering all conditions, 0.5% maltodextrin addition was the best for stabilizing SAP powders along storage time.

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&amp / #8805 / 2.5 mM. The highest SAP activities with asparagine, aspartic acid, lysine, threonine, isoleucine and methionine werefound to be 1.89-, 1.87-, 1.61-, 1.48-, 1.4-, and 1.4-fold higher than the reference medium activity, respectively, when the concentration of each amino acid was CAA=0.25 mM. The product and by-product distributions in defined and complex media in SAP production were also analyzed and compared in order to obtain a depth in-sight on functioning of the metabolic reaction network. The highest SAP activity in complex medium was found to be 3&ndash / fold higher than defined medium activity, while, specific SAP production rate was 1.2- fold higher. The highest cell concentration in complex medium (CX= 14.3 g/dm-3) was 8.1-fold higher than that obtained in defined medium (CX= 1.75 g/dm-3). In both media, oxaloacetic acid was observed extracellularly and intracellularly. In complex medium there was also succinic acid in the extracellular medium indicating that the operation of TCA cycle was insufficient. In both media serine, valine and glycine were observed neither in the extracellular nor in the intracellular media indicating that the synthesis of these amino acids can be a secondary rate limiting step. In defined medium asparagine was present neither in the cell nor in fermentation broth whereas, methionine was observed in the cell in high amounts, probably due to the lower flux values towards asparagine. Thus, in defined medium the synthesis of asparagine can also be a potential bottleneck in SAP production in defined medium.