Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens Biovar I, a thiamine pyrophosphate (ThDP) dependent enzyme, catalyzes the enzymatic kinetic resolution of racemates by C-C bond cleavage and concomitant C-C bond formation. In this study, benzaldehyde lyase gene from Pseudomonas fluorescens Biovar I was cloned into Pichia pastoris, with the aim of the extracellular production of the enzyme. For this purpose, firstly, PCR amplified bal gene was cloned into an integration vector pPICZalphaA. Thereafter the recombinant plasmid pPICZalphaA::bal was transformed into P.pastoris. Extracellular benzaldehyde lyase enzyme was expressed under the control of the strong AOX promoter and the secretion of the enzyme in the fermentation medium was achieved by means of S. cerevisiae alpha factor signal sequence. The recombinant cells were grown for 48-72 hours in solid medium then the cells inoculated in glycerol containing medium. After being separated by centrifugation cells were transferred into methanol containing production medium. In methanol containing medium cells were grown for 72 h. Starting from t=24 h methanol was added to medium as an inducer of AOX promoter and the carbon source in order to produce BAL in every 24 hour. SDS-PAGE analyses illustrated that extracellular benzaldehyde lyase enzyme produced by the recombinant P.pastoris strain had the size of 59 kDa, which is the size of benzaldehyde lyase monomer. FPLC analysis showed that concentration of the tetrameric form of benzaldehyde lyase enzyme, active form, was much less than the monomeric form of the enzyme indicating that the enzyme produced by recombinant P.pastoris mostly could not fold into multimeric form in the fermentation medium.

In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxygen transfer rate, volumetric and specific oxygen uptake rates, mass transfer and biochemical reaction resistances / moreover, the variation in product and by-product distribution, specific substrate uptake rates, yield and maintenance coefficient were investigated in the pilot scale batch bioreactor at QO/VR = 0.5 vvm and agitation rates of N= 250, 500, 625, and 750 min-1, and dissolved oxygen levels DO= 20%, 40% conditions, while medium components were CGlucose= 8.0 kg m-3, C(NH4)2HPO4= 5.0 kg m-3 and salt solution at controlled pHc=7.2. The highest cell concentration and benzaldehyde lyase activity were obtained at DO=40% condition as 3.0 kg m-3 and A=1095 Ucm-3, respectively. v Then a mathematical model was proposed to estimate benzaldehyde lyase activity as function of time, agitation rate, cell concentration, dissolved oxygen concentration, and by-product concentration with reasonable accuracy.

This study focuses on the molasses based complex medium design for benzaldehyde lyase production by recombinant E. coli BL21 and development of a feeding strategy based on the designed complex medium. For this purpose, firstly, the effects of molasses were investigated in laboratory scale bioreactors. As E. coli BL21 was not able to utilize sucrose, molasses was pretreated and hydrolyzed to fructose and glucose. Thereafter, effect of pretreated molasses concentration was investigated in the range of 16 to 56 kg m-3 by batch-bioreactor experiments / and the highest cell concentration and benzaldehyde lyase activity were obtained as CX=5.3 kg m-3 and A=1617 U cm-3, respectively, in the medium containing 7.5 kg m-3 glucose and 7.5 kg m-3 fructose. Then, different feeding strategies were developed to produce efficient cells with high concentration and BAL activity. In the first strategy, after 10 hours of batch-cultivation with molasses based medium having 7.5 kg m-3 glucose and 7.5 kg m-3 fructose concentration, based on the airflow rate, pretreated molasses was fed to the system. When air flow rate decreased considerably, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3. At the end of the process, the highest cell concentration obtained was CX=7.4 kg m-3. The maximum activity was reached at 20th hour as A=2360 U cm-3. On the other hand, as air flow variation only demonstrated the absence of glucose not fructose, a second strategy, based on the detection of the fructose and glucose concentrations during the process, was applied. In this strategy when glucose and fructose were depleted, fed was given to the system that results in increase in glucose and fructose concentration in the medium to 2.5 kg m-3 / and the highest BAL activity was obtained as 2370 U cm-3 at t= 26 h where the cell concentration was 7.5 kg m-3. At the last strategy, when glucose and fructose were depleted, fed was given to the system that results in increase in CGlucose=1.5 kg m-3 and CFructose=1.5 kg m-3 in the production medium to decrease the accumulation of acetic acid. By this strategy highest cell concentration was obtained as 8.04 kg m-3 at t=24 h and the highest BAL activity was 2315 U cm-3. These strategies could be accepted having the same BAL activity with little distinctions. However, cell concentration of the last one was higher than others and also the lowest amount of carbon source was used. Thus, last one could be chosen as the most favorable strategy.

In this study, the effects of pH on therapeutically important protein, recombinant human erythropoietin (rhuEPO), production by Pichia pastoris was investigated at pH=4.0, 4.5, 5.0, 5.5 and 6.0. rHuEPO production was started by methanol induction in fed-batch mode. The highest cell concentration was obtained at pH=4.5 as 81.4 g L-1. The co-substrate substrate sorbitol, which was added batch-wise, was consumed at t=15 h of the operations at pH=4.0, 4.5 and 5.0. However as the pH increases above pH=5.0 the sorbitol consumption rate decreases. The highest rHuEPO concentration was achieved at pH=4.5 as 0.158 g L-1 which was 1.43-, 1.24-, 1.95- and 1.23-fold higher than those obtained at pH=4.0, 5.0, 5.5, and 6.0, respectively. Also at pH=4.5 overall cell yield on substrate was 0.51 g g-1 and overall rHuEPO yield on substrate was 1.45 mg g-1. rHuEPO concentration was decreased in the last 3-6 hour of the operation due to proteolysis. Therefore extracellular protease concentrations in the medium were determined. As expected, since the investigated pH range was acidic, the amount of acidic proteases was found to be higher than neutral and basic proteases. Furthermore the total protease concentration increased linearly in the fermentation broth, having close values at different pH values. Thus, pH did not have a significant effect on extracellular protease activity. Alcohol oxidase (AOX) activities showed similar behavior at different pH. The highest specific AOX activity was attained at pH=4.5, at which the highest rHuEPO concentration was achieved, as 110.1 U g-1 CDW. Keywords:

In this study, the aim was to investigate the effects of pH on therapeutically important protein, recombinant human growth hormone (rhGH), production by Pichia pastoris considering the expression levels of regulatory genes. In this frame, firstly the host microorganism was selected between two different methanol utilization phenotypes of P. pastoris, Mut+ and MutS on media containing glycerol/methanol or sorbitol/methanol. The highest rhGH production, 120 g L-1, and hGH gene expression, 9.84x109 copies mg-1 CDW, were achieved in the medium containing 30 g L-1 sorbitol and 1% (v/v) methanol by P. pastoris hGH-Mut+ strain. Thereafter, effects of pH on rhGH production and stability were investigated in laboratory scale bioreactors. RhGH was more stable at pH 5.0. Throughout the production, it is seen that medium of pH decreased. Thereafter, effects of pH on rhGH were investigated in pH controlled pilot-scale bioreactor. In addition to rhGH concentration, AOX intracellular enzyme activity, extracellular proteases concentrations / expression levels of hGH, AOX, pep4, prb1 and prc1 genes were determined. The highest cell concentration was obtained as 53 g L-1 at pH 6.0 but hGH concentration was found as 24 mg L-1 at t=24 h. The highest rhGH concentration was obtained as 271 g L-1 with 42 g L-1 cell density at pH 5.0 in medium containing sorbitol at t=24 h. At this condition, the overall product and cell yield on total substrate were found as 2.08 mg g-1 and 0.15 g g-1. Furthermore, the highest expression levels of hGH and AOX were attained at pH 5.0. Moreover, by keeping pH at 5.0, expression levels of three types of vacuolar proteases were minimized.

Hepatitis B virus (HBV), which belongs to the family Hepadnaviridae, is responsible for acute and chronic hepatitis. The vaccines presently used to immunize patients against HBV are recombinant subunit vaccines consisting of viral surface antigens (S protein). However, they are expensive and their use is limited in poor countries. For that reason, HBV remains an important worldwide health problem. Of the 2 billion people who have been infected with the HBV, more than 350 million have chronic (lifelong) infections, who face increased risk of developing cirrhosis and hepatocellular carcinoma. In this study, high-level expression of recombinant Hepatitis B surface Antigen (rHBsAg), PreS2-S was achieved in the methylotrophic yeast, Pichia pastoris. For this aim, a single copy of HBV M gene (PreS2-S) was inserted at the downstream of the alcohol oxidase (AOX1) promoter of the pPICZA vector. rHBsAg protein could then be expressed intracellularly by induction with methanol. High cell density fermentation was followed by chromatographic separation to obtain pure rHBsAg. Humoral response after immunization with the purified protein was observed in mice using commercial Hepatitis B surface antigen kits. It was verified by the atomic force microscopy that rHBsAg has been produced in the desired conformation.

In this study, the aim was to achieve high concentration of recombinant human growth hormone (rhGH) production by recombinant Pichia pastoris by investigating the effects of various operation parameters and to determine the suitable peptide ligand sequence that shows affinity and specificity to hGH. In this context, firstly the effect of temperature and Tween-20/80 addition on production and cell growth were investigated. While at T=30 and 32&deg / C, there was no difference, at 27 and 25&deg / C cell growth slowed down and production decreased significantly. The addition of Tween-20/80 in existence of co-substrate sorbitol did not affect the bioprocess while in absence of sorbitol Tween alone did not show the same positive effect on product formation and cell growth. Thereafter at T=30&deg / C, without addition of Tween, three sets of pilot scale bioreactor experiments were performed. In the first set, the effect of methanol feeding rate on bioprocess characteristics were investigated at the specific growth rates of &mu / =0.02, 0.03 and 0.04 h-1. While the highest cell concentration was achieved at &mu / =0.04 h-1, the highest rhGH concentration was achieved at &mu / =0.03 h-1. Secondly, conducting methanol feeding at &mu / =0.03 h-1, pH=5.5 experiment was conducted. The highest cell concentration, 45 g L-1 and maximum rhGH concentration 0.25 g L-1 were achieved at t=18 h of the process. Finally, the effect of batch sorbitol feeding on bioprocess was observed by the addition of 50 g L-1 sorbitol at t=0, 14 and 31 h of the production phase. It was shown that sorbitol addition to the medium increased process duration / hence cells enter stationary phase after a longer production phase. However, the protease concentration continued increasing with respect to time and at the end of the process reached twice the concentration it was obtained with single sorbitol addition case decreasing the rhGH concentration. In selection of the peptide sequence that shows affinity towards hGH, phage display method was conducted. Additionally the sequences from literature and computational design were used as alternatives. The interaction between these peptides and hGH was investigated by isothermal titration calorimetry and surface plasmon resonance.

In this study, regulatory gene effects on recombinant human growth hormone (rhGH) production by Bacillus subtilis were investigated. For this purpose, firstly Bacillus strains, which are deficient in abrB, aprE, degQ, degS, degU, scoC, sinI, sinR, and spo0A genes, were selected according to the regulatory gene network of aprE gene (serine alkaline protease gene of B. subtilis) since due to the degQ promoter and the pre-signal sequence of subC gene cloned in front of the hGH gene, hGH is produced by mimicking the serine alkaline protease synthesis. R-Bacillus strains were constructed by transformation of pMK4::pre(subC)::hGH plasmid to the selected strains. Thereafter, by the laboratory scale experiments, strains having the highest hGH production capacity were determined as scoC, aprE, sinR, and degU knockout strains. Using these strains, fermentation experiments were carried out in pilot-scale bioreactor in defined medium. Effect of pH control was also investigated and the highest cell and hGH concentration was obtained by scoC knockout strain in pH controlled operation as 1.62 kg m-3 and 126 g m-3, respectively. By this strain, the overall product and cell yield on total substrate were found as 16.12 g kg-1 and 0.15 g g-1, respectively. Furthermore, the highest total protease activity was attained by degU knockout strain as 65 U cm-3. On the other hand, maximum total organic acid secretion was determined as 1.31 kg m-3 in aprE knockout strain.

Pyrococcus furiosus extracellular &alpha / -amylase is a hyperthermostable glucosyl hydrolyzing enzyme which shows unique biochemical properties that may have impact on improving starch hydrolysis process / however, it is insignificantly expressed in its native archaeal host. In this study, it was aimed to express the P. furiosus extracellular alpha-amylase (PFA) in Pichia pastoris, which is a well-recognized overexpression host used in production of heterologous proteins. In this context, first, P. furiosus was grown under anaerobic conditions in capped bottles for t= 12 h at T=90&deg / C and then its genomic DNA was isolated. PFA coding cDNA frame was amplified using two specifically designed oligonucleotides and cloned into pPICZ&alpha / A expression vector. Then wild type P. pastoris X-33 cells were transfected with pPICZ&alpha / A::PFA construct. In shake flask production medium, existence of recombinant PFA activity was tested and biochemical characterization of the recombinant product was done. This was the first time PFA is expressed in an eukaryotic host. Optimum working temperature and pH of the rPFA were found to be 95 &deg / C and within the range of 4.5-6.5, respectively. rPFA is independent to metal ions and inhibition by production medium of P. pastoris was observed, in presence of divalent metal ions. Although Saccharomyces cerevisiae &alpha / -factor secretion signal was fused to the N terminal of rPFA, minute amount of extracellular secretion was detected but the majority of the enzymatic activity remained in the intracellular medium. The best producer strain was selected by measuring &alpha / -amylase activity in cell extracts by DNS method. Effects of pH on cell growth and recombinant protein production were determined by shake flask experiments and maximum of 4800 U/l rPFA was detected with 7.30 g/l wet cell density in pH=6 buffered medium. In order to achieve higher rPFA production, two bioreactor experiments were designed at two different pH operation conditions, namely pH=4 and pH=5, in a working volume of 1 L. The dissolved oxygen tension was kept over 20% and predetermined exponential methanol feeding strategy was employed in order to fix specific cell growth rate, &micro / , at 0.03 h-1. At pH=4 operation, maximum of 73,400 U/l &alpha / -amylase activity was detected at the t=27 h of production phase when the wet cell density was 209 g/l.

In this study, recombinant human growth hormone (rhGH) production by Pichia pastoris-Mut+ strain was improved by designing feeding strategies which were applied in the production phase of the bioreactor operations. During the bio-reactor experiments the cell growth, sorbitol and methanol consumptions, recom-binant hGH production, alcohol oxidase (AOX) activity, the by-products protease and organic acid concentrations were followed and analyzed. In this context, in the first part of the study, three bioreactor operations were designed and per-formed. In general, the designed strategies are fundamentally based on simulta-neous feeding of the two substrates starting at t=0 h of the production phase, i.e., batch-wise 50 gL-1sorbitol feeding, together with fed-batch methanol feeding with a specific growth rate of &mu / 0=0.03 h-1 or &mu / 0=0.04 h-1, and fed-batch sorbitol feeding with a specific growth rate of &mu / 0=0.025h-1 which was calculated based on the specific consumption rate qS=0.152 g g-1h-1 of sorbitol. Consequently, sorbitol concentration was kept constant at 50 gL-1 within t=0-15h of the production phase / where, sorbitol feeding was terminated at t=15h. Amongst, in the first strategy (SSM1), methanol was fed to the system with the specific growth rate of &mu / 0=0.03 h-1, and the H+ concentration (pH) in the bioreactor was kept constant at pH=5.0. In the second strategy (SSM2), pH was kept constant at 5.5 until t=24h of the induction phase (production phase), thereafter, was reduced to pH= 5.0 / where methanol was fed to the bioreactor with the specific growth rate of &mu / 0=0.03 h-1. In the third strategy (SSM3), methanol was fed with the specific growth rate of &mu / 0=0.04 h-1, and the pH in the bioreactor was kept constant at pH 5.0. The highest rhGH production and cell concentration were achieved in the first strategy SSM1 as CrhGH=640 mg L-1 and CX=105.3 g L-1, and the overall cell and product yields on total substrate were calculated as YX/S =0.21 g g-1 and YCrhGH/S =1.83 mg g-1. In the second part of this study the two-substrates sorbitol and methanol were fed simultaneously in a solution compose of 1.37 mol sorbitol and 6.21 mol methanol in 13.88 mol water, which is named as SM. In this strategy (SM), the two-substrate solution was fed to the medium with the specific growth rate of &mu / 0=0.03 h-1 on sorbitol until t=30h / thereafter, only methanol was fed to the bio-reactor with the specific growth rate of &mu / 0=0.03 h-1. The highest cell and rhGH concentrations obtained in SM were, respectively, Cx=104.7 g L-1 and CrhGH=124 mg L-1 / and the overall cell and product yields on the total substrate were calcu-lated as YX/S=0.21 g g-1 and YCrhGH/S=0.39 mg g-1. Although the highest cell con-centration obtained at SM is close to that of the SSM1, the rhGH concentration obtained at SM is 5.2-fold lower than that of the strategy SSM1.