Person: PİNAR, ORKUN
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PİNAR
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ORKUN
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Publication Open Access A two-step purification platform for efficient removal of Fab-related impurities: A case study for Ranibizumab(2023-11-01) PİNAR, ORKUN; SARIYAR AKBULUT, BERNA; KAZAN, DİLEK; Tatli O., Oz Y., Dingiloglu B., Yalcinkaya D., Basturk E., Korkmaz M., Akbulut L., Hatipoglu D., Kirmacoglu C., Akgun B., et al.Antibodies (mAbs) and antibody fragments (Fabs) constitute one of the largest and most rapidly expanding groups of protein pharmaceuticals. In particular, antibody fragments have certain advantages over mAbs in some therapeutic settings. However, due to their greater chemical diversity, they are more challenging to purify for large-scale production using a standard purification platform. Besides, the removal of Fab-related byproducts poses a difficult purification challenge. Alternative Fab purification platforms could expedite their commercialization and reduce the cost and time invested. Accordingly, we employed a strong cation exchanger using a pH-based, highly linear gradient elution mode following Protein L affinity purification and developed a robust two-step purification platform for an antibody fragment. The optimized pH gradient elution conditions were determined on the basis of purity level, yield, and the abundance of Fab-related impurities, particularly free light chain. The purified Fab molecule Ranibizumab possessed a high degree of similarity to its originator Lucentis. The developed purification platform highly intensified the process and provided successful clearance of formulated Fab- and process-related impurities (∼98 %) with an overall process recovery of 50 % and, thus, might be a new option for Fab purification for both academic and industrial purposes.Publication Open Access A preliminary study on purification and characterization of lipase(s) produced by cryptococcus diffluens D44(2023-03-01) PİNAR, ORKUN; Büyük E., Pinar O.In the present work, preliminary purification, and characterization of lipases from Cryptococcus diffluens D44, which was isolated from petroleum sludge, were performed. In the purification steps, subsequential to acetone precipitation, lipases from C. diffluens D44 were purified by DEAE Sepharose resulting in two different peaks, named Lip1 and Lip4. Sephadex G-100 size-exclusion chromatography was also performed for further purification of Lip1 and Lip4 and resulted in three different lipases as Lip1-1 (1.0 purification fold with 2.4% recovery), Lip1-2 (0.8 purification fold with 7.2% recovery), and Lip4-1 (1.2 purification fold with 4.5% recovery). As a result of characterization studies of these three lipases resulting from different peaks, optimum temperatures were found as 60 °C, 65 °C, and 65 °C for Lip1-1, Lip1-2, and Lip4-1, respectively. Furthermore, thermal stability studies were conducted at 50 °C, 60 °C, and 70 °C, and lipases of C. diffluens D44 maintained over 70% of their initial activity at 50 °C. The optimum pH for Lip1-1 and Lip1-2 was pH 9.0 although pH 5.0 was for Lip4-1. Considering the organic solvent effect on lipase activity, 10% methanol enhanced the relative activity of Lip1-1 and Lip4-1 while 10% ethanol caused a decrease in the relative activity of lipases except for Lip1-2. According to the indicated features based on the results, these different lipases from C. diffluens D44 could be promising candidates for industrial and biotechnological applications. To the best of our knowledge, this is the first study on the purification of lipases from C. diffluens D44Publication Open Access Determination of the Significance of the Most Effective Nutrients on Lipase Production from Cryptococcus albidus D24(2022-06-01) PİNAR, ORKUN; KAZAN, DİLEK; Uras, Abdussamed; Uras A., Pinar O., Kazan D.In this study, the optimization of the medium components used for the production of lipase enzyme from Cryptococcus albidus D24 was performed using the Plackett-Burman statistical design method (PBD), and the most important nutrients affecting the production of lipase enzyme from D24 strain were determined as the first step. According to PBD, the highest lipase activity (19.34 U/ml/min) was obtained with medium including Tween 80 (X2) 2.5% (v/v), and (g/L) Peptone (X4) 8.0, Yeast Extract (X6) 7.5, Beef Extract (X7) 7.5, Malt Extract (X8) 7.5, NH4Cl (X9) 6.0, NaNO3 (X10) 1.5, (NH4)NO3 (X12) 6.0, (NH4)HCO3(X13) 6.0, MgSO4.7H2O (X15) 1.0, and KH2PO4 (X16) 2.0 at the end of 144 h cultivation. Regarding the concentration effect (CE) values obtained from PBD, NH4Cl (CE=7.1587), olive oil (CE=3.5544), (NH4)HCO3 (CE=3.0747), and tryptone (CE=2.1427) were evaluated as the more effective nutrients among the sixteen compounds studied. After that, the optimum concentrations of these effective compounds were experimented with Response Surface Methodology (RSM). Experimental results showed that the medium containing olive oil (X3) 1.5% (v/v), and (g/L) tryptone (X5) 3.0, NH4Cl (X9) 7.5, and (NH4)HCO3 (X13) yielded maximum lipase activity (12.03 U/ml/min) compared to other studied compounds. Although the maximum lipase activity obtained with RSM methodology was lower than that obtained by PBD, the cost of the nutrients used to produce one-unit enzyme is 0.104 Euro in the PBD, while only 0.0277 Euro is spent in RSM. In other words, the production of lipase using compounds coded X3, X5, X9, and X13 provides a cost-effective process.Publication Open Access Investigation of the Physiology of the Obligate Alkaliphilic Bacillus marmarensis GMBE 72(T) Considering Its Alkaline Adaptation Mechanism for Poly(3-hydroxybutyrate) Synthesis(MDPI, 2021-02-23) KAZAN, DİLEK; Atakav, Yagmur; Pinar, Orkun; Kazan, DilekThe novel extreme obligate alkaliphilic Bacillus marmarensis DSM 21297 is known to produce polyhydroxybutyrate (PHB). However, the detailed mechanism of PHB synthesis in B. marmarensis is still unknown. Here, we investigated which metabolic pathways and metabolic enzymes are responsible for PHB synthesis in order to understand the regulatory pathway and optimize PHB synthesis in B. marmarensis. In accordance with the fact that beta-galactosidase, 3-hydroxyacyl-CoA dehydrogenase, and Enoyl-CoA hydratase together with acyl-CoA dehydrogenase and lipase were annotated in B. marmarensis according to the RAST server, we used glucose, lactose, and olive oil to understand the preferred metabolic pathway for the PHB synthesis. It was found that B. marmarensis produces PHB from glucose, lactose, and olive oil. However, the highest PHB titer and the highest amount of PHB synthesized per dry cell mass (Y-P/X) were achieved in the presence of lactose, as compared to glucose and olive oil. Additionally, in the absence of peptone, the amount of PHB synthesized is reduced for each carbon source. Interestingly, none of the carbon sources studied yielded an efficient PHB synthesis, and supplementation of the medium with potassium ions did not enhance PHB synthesis. According to these experimental results and the presence of annotated metabolic enzymes based on the RAST server, PHB accumulation in the cells of B. marmarensis could be improved by the level of the expression of 3-hydroxybutyryl-CoA dehydrogenase (1.1.1.157), which increases the production of NADPH. Additionally, the accumulation of 3-hydroxyacyl-CoA could enhance the production of PHB in B. marmarensis in the presence of fatty acids. To our knowledge, this is the first report investigating the regulatory system involved in the control of PHB metabolism of B. marmarensis.