Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity. In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions. <b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity

Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl transfer reaction between a &gamma / -carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular microbial protransglutaminase (pro-MTG) producing recombinant Pichia pastoris strains by using genetic engineering techniques. In this context first,protransglutaminase gene (pro-mtg) from Streptomyces mobaraensis was amplified by PCR both for intracellular and extracellular constructs using proper primers then they were cloned into the pPICZ&alpha / -A expression vectors, separately. Both intracellular (pPICZ&alpha / A::pro-mtgintra) and extracellular (pPICZ&alpha / A::pro-mtgextra) constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Pichia pastoris X33 cells were transfected by linear pPICZ&alpha / A::pro-mtgintra and pPICZ&alpha / A::pro-mtgextra, separately and plasmids were integrated into the Pichia pastoris X33 genome at AOX1 locus. After constructing the recombinant P. pastoris strains, batch shaker bioreactor experiments were performed for each recombinant cell and the best producing strains were selected according to Dot blot and SDS-PAGE analyses. The selected recombinant P. pastoris strains, carrying pPICZ&alpha / A::promtgextra gene and pPICZ&alpha / A::pro-mtgintra gene in their genome were named as E8 and I1, respectively. Afterwards, a controlled pilot scale bioreactor experiment in a working volume of 1 L was performed with E8 clone and produced pro-MTG was activated by Dispase I. The variations in the recombinant MTG activity, cell concentration, total protease activity, AOX activity and organic acid concentrations throughout the bioprocess were analyzed and specific growth rates, specific consumption rates and yield coefficients were calculated regarding to measured data. Maximum MTG activity was obtained as 4448 U L- 1 and the maximum cell concentration was measured as 74.1 g L-1 at t=36 h of the bioprocess. In this study, an active transglutaminase enzyme was produced extracellularly by P. pastoris for the first time and the third highest extracellular MTG activity was achieved with E8 clone.

In this study, it was aimed to investigate the effects of different co-carbon sources on therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by Pichia pastoris by designing feeding strategies which were applied in the production phase of the bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions, recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the by-products organic acid concentrations were analyzed. In this context, firstly, laboratory scale air filtered shake bioreactor experiments were performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial concentration of co-substrates was found more affordable and appropriate for cell concentration and recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the production phase together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system with different feeding strategies together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1 mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy (MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h, containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1 at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and YP/St=2.23 mg g-1 in the highest rHuEPO production case.

In this study, the effects of bioprocess operation parameters on recombinant human growth hormone (rhGH) production by P. pastoris were systematically investigated. In this frame, first, for the extracellular expression and purification of human growth hormone by recombinant P. pastoris the cDNA of hGH, fused with a polyhistidine tag and also fused with a target site for the Factor Xa protease in which cleavage produces a mature N- and C- termini of rhGH, was cloned into pPICZ&amp / #945 / A plasmid and the constructed system within the plasmid, pPICZ&amp / #945 / A::hGH, was integrated to AOX1 locus of P. pastoris and expressed under alcohol oxidase promoter which is induced by methanol. With dot-blot analysis, the appropriate two strains producing human growth hormone at high levels and having different methanol utilization phenotype (Mut+ and Muts) were chosen among the other transformants. Then, the effects of methanol concentrations on the expression of rhGH and cell growth were analyzed and both of the phenotypes were compared in defined and complex media in laboratory scale air filtered shake bioreactors. The highest rhGH concentration for Mut+ and MutS, was found as 0.052 kg m-3 and 0.16 kg m-3, respectively, at 2 %(v/v) methanol concentration in complex medium. When methanol was used as the sole carbon source in defined medium, Muts phenotype had very low specific growth rate on methanol due to the intrinsic characteristics of it, therefore detectable rhGH was not observed, on the other hand, optimum rhGH concentration produced by Mut+ strain was found as 0.032 kg m-3 at 3% (v/v) methanol concentration in defined medium. In mixed system (glycerol/methanol) which is also defined, when the optimum glycerol concentration, 30 kg m-3, was used, Muts produced the highest rhGH, 0.110 kg m-3, at 1% (v/v) methanol concentration and any increase in methanol concentration resulted in lower rhGH production, on the other hand, Mut+ strain produced 0.060 kg m-3 rhGH at 4% (v/v) methanol concentration, which indicated that higher rhGH production capacity of Mut+ strain was obtained at high methanol concentrations. Using the designed defined medium for Mut+ phenotype where methanol was used as the sole carbon source with an optimum concentration of 3% (v/v), the effects of oxygen transfer on rhGH production, by-product formation, and cell growth, oxygen transfer and fermentation characteristics were investigated by using pilot scale bioreactor. Oxygen transfer effects on rhGH production were investigated at QO/VR=0.5 vvm / N=250, 500, 625, 750 min-1 conditions. The variations in rhGH , cell, amino acid and organic acid concentrations with the cell cultivation time, specific cell growth rate, the oxygen uptake rate, the liquid phase coefficient by using the dynamic method, maintenance coefficient for oxygen and yield coefficients were determined. The highest rhGH concentration was obtained at 0.5 vvm, 500 min-1 condition as 0.023 kg m-3 with 5.37 kg m-3 cell density.

Extracellular endo-1,4-b-mannanase (EC 3.2.1.78) gene of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was cloned and transformed into Aspergillus sojae (ATCC 11906) and Pichia pastoris GS115. High level of expression was achieved in both expression systems. Attempts to produce heterologous mannanase in Arabidopsis thaliana, suitable for large scale production, were not successful. Comparison of the expression levels of heterologous mannanase reveals that A. sojae is a better expression system than P. pastoris with respect to extracellular mannanase activity. The production of mannanase in A. sojae (AsT1) after 3 days of incubation reached 204 U/ml in YpSs containing 1 % glucose. In P. pastoris (PpT1), highest production was observed after 10 hrs of induction with methanol (61 U/ml). Expressed enzymes were purified and analyzed. Both enzymes have specific activity c. 349 U/mg protein with pH and temperature optimum of c. 4.5 and c. 60 &deg / C for mannanases from AsT1 and c. 5.2-5.6 and c. 45 &deg / C for mannanases from PpT1. A truncated form of mannanase (MAN-S) deleted at amino acids from P291 to P368, which still displayed hydrolytic activity was also isolated and characterized. MAN-S has pH and temperature optimum of c. 6.5-8.0 and c. 60 &deg / C. During incubation of the mannanase on locust bean gum, transglycosylation reactions, in which longer or rare prebiotic oligosaccharides could be produced catalyzed by glycolysis, was detected. The products of hydrolytic activity of the enzyme on various carbohydrates were analyzed by PACE and MALDI-TOF. Accordingly, hexamannose and smaller oligosaccharides were characterized.

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.

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.