Browsing by Author "Unver, Yagmur"

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Affinity-Based and In a Single Step Purification of Recombinant Horseradish Peroxidase A2a Isoenzyme Produced by Pichia Pastoris
(Springer, 2023) Acar, Melek; Abul, Nurgul; Yildiz, Seyda; Taskesenligil, Ezgi Dag; Gerni, Serpil; Unver, Yagmur; Ozdemir, Hasan
Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZ alpha C. So, obtained pPICZ alpha C-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.
Combination of Magnetic Hyperthermia and Gene Therapy for Breast Cancer
(Springer, 2025) Solak, Kubra; Arslan, Seyda Yildiz; Acar, Melek; Turhan, Fatma; Unver, Yagmur; Mavi, Ahmet
This study presented a novel breast cancer therapy model that uses magnetic field-controlled heating to trigger gene expression in cancer cells. We created silica- and amine-modified superparamagnetic nanoparticles (MSNP-NH2) to carry genes and release heat under an alternating current (AC) magnetic field. The heat-inducible expression plasmid (pHSP-Azu) was designed to encode anti-cancer azurin and was delivered by magnetofection. MCF-7 cells demonstrated over 93% cell viability and 12% transfection efficiency when exposed to 75 mu g/ml of MSNP-NH2, 3 mu g of DNA, and PEI at a 0.75 PEI/DNA ratio (w: w), unlike non-tumorigenic cells (MCF-10 A). Magnetic hyperthermia (MHT) increased azurin expression by heat induction, leading to cell death in dual ways. The combination of MHT and heat-regulated azurin expression induced cell death, specifically in cancer cells, while having negligible effects on MCF-10 A cells. The proposed strategy clearly shows that simultaneous use of MHT and MHT-induced azurin gene expression may selectively target and kill cancer cells, offering a promising direction for cancer therapy.
Invertase Production and Molasses Decolourization by Cold-Adapted Filamentous Fungus Cladosporium Herbarum Er-25 in Non-Sterile Molasses Medium
(Elsevier, 2016) Taskin, Mesut; Ortucu, Serkan; Unver, Yagmur; Tasar, Ozden Canli; Ozdemir, Mustafa; Kaymak, Haluk Caglar
This study was undertaken to remove the coloring compounds of molasses as well as produce extracellular (exo) invertase in sterile and non-sterile molasses medium by using cold-adapted filamentous fungus Cladosporium herbarum ER-25. It was determined that a combination of low culture pH (5.5), temperature (20 degrees C) and high molasses concentration (6%) could completely prevent undesired bacterial contamination during the cultivation of C. herbarum ER-25. Under the optimized non-sterile culture conditions, the maximum invertase activity (36.1 U/mL) was attained after 72 h. On the other hand, the fungus could remove toxical dark brown pigments (melanoidins) in non-sterilized molasses medium through biodegradation and bioadsorption mechanisms. A color removal rate of 64.8% in non-sterile medium could be achieved at the end of 144-h cultivation period. It was found that lac case and manganese peroxidase were responsible for biodegradation. No ligninase activity was detected for the fungus during the cultivation. Maximum laccase (4.6 U/mL) and manganese peroxidase (3.5 U/mL) activities could be reached after 120 h. Higher invertase activity and color removal rate were achieved in non-sterilized medium compared to sterilized one. This is the first report on invertase production from cold-adapted microorganisms under non-sterile culture conditions. As an additional contribution, use of cold-adapted fungi for molasses decolourization was investigated for the first time in the present study. (C) 2016 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
L-Lactic Acid Production by Rhizopus Oryzae Mbg-10 Using Starch-Rich Waste Loquat Kernels as Substrate
(Wiley-V C H Verlag GmbH, 2013) Taskin, Mesut; Ortucu, Serkan; Unver, Yagmur; Arslan, Nazli Pinar; Algur, Omer Faruk; Saghafian, Amir
The objective of this work was to perform production of L-lactic acid from starch-rich waste loquat kernels by newly isolated Rhizopus oryzae MBG-10 fungus. Loquat kernel flour (LKF) was used as substrate (mainly as carbon source). The most favorable conditions for L-lactic acid production were LKF concentration of 80g/L, CaCO3 concentration of 20g/L, ammonium sulfate concentration of 3g/L and incubation time of 108h. Under these conditions, L-lactic acid and biomass concentrations were 45.4 and 8.2g/L, respectively, and -amylase activity was 81.6U/mL. No significant pH changes were observed in the medium thanks to the buffering capacity of LKF. L-lactic acid could be produced in a single-stage from starch-rich LKF without prior saccharification by the fungus with high amylolytic enzyme activity. This is the first report on use of waste loquat kernels as a lactic acid production substrate.
Lipase Production with Free and Immobilized Cells of Cold-Adapted Yeast Rhodotorula Glutinis HL25
(Elsevier Science BV, 2016) Taskin, Mesut; Ucar, Muhammed Hanifi; Unver, Yagmur; Kara, Ayse Aydan; Ozdemir, Mustafa; Ortucu, Serkan
This study was undertaken to produce the lipase by free and immobilized cells of cold-adapted yeast Rhodotorula glutinis HL25 using waste frying oils as substrate. The optimization of culture parameters was performed using traditional one-factor-at-a-time protocol. The temperature 20 degrees C and initial pH 6.0 were optimal for lipase production by both free and immobilized cells. An inoculum size of 40 mL/L for free cells and beads number of 150 g/L for immobilized cells were optimal for lipase production. Optimal waste frying oil concentration and incubation time were 30 mL/L and 84 h for free cells but 40 mL/L and 72 h for immobilized cells, respectively. The maximum increases for free and immobilized cells were achieved at the Triton X-100 concentrations of 5 and 7.5 mL/L, respectively. The maximum lipase activities were determined as 54.4 and 75.2 U/L for free and immobilized cells, respectively. Immobilized cells could be used in five successive reaction cycles without any loss in the maximum activity. Immobilized cells could retained about 70% of their maximum activity by the end of the cycle 10. This is the first attempt on lipase production potential of a cold-adapted strain of the yeast R. glutinis. Furthermore, lipase production using immobilized cells of cold-adapted yeasts was investigated for the first time. (C) 2016 Elsevier Ltd. All rights reserved.
Nitric Oxide: A Novel Inducer for Enhancement of Microbial Lipase Production
(Springer, 2016) Taskin, Mesut; Unver, Yagmur; Yildiz, Melike; Ortucu, Serkan; Askin, Hakan
The purpose of this study was to elucidate whether exogenous nitric oxide (NO) has a potential beneficial effect on lipase production capacity of some microorganisms. Sodium nitroprusside (SNP) was used as an exogenous NO donor in production medium. In comparison with the control (0 nM SNP), SNP concentrations from 10 to 100 nM induced lipase production in mesophilic bacterium Bacillus subtilis and cold-adapted yeast Yarrowia lipolytica. Especially, the maximum lipase activities for Y. lipolytica (81.2 U/L) and B. subtilis (74.5 U/L) were attained at 30 and 50 nM SNP concentrations, respectively. When compared to the control, the optimal SNP concentrations resulted in about 5.14 and 2.27-fold increases in lipase activities of B. subtilis and Y. lipolytica, respectively. Besides, it was found that the optimal SNP concentrations provided shorter incubation periods for lipase production. Conversely, no significant positive effect of exogenous NO on lipase production was determined for thermophilic bacterium Geobacillus stearothermophilus. This study showed for the first time that exogenous NO could be used as an inducer in the production of microbial lipases.
Protease Production by Free and Immobilized Cells of the Cold-Adapted Yeast Cryptococcus victoriae CA-8
(Taylor & Francis Ltd, 2015) Unver, Yagmur; Yildiz, Melike; Taskin, Mesut; Arslan, Nazli Pinar; Ortucu, Serkan
The present study was performed to produce the protease using free and immobilized cells of locally isolated cold-adapted psychrotolerant yeast Cryptococcus victoriae CA-8. Cell immobilization was performed using sodium alginate as entrapping agent. The best conditions for enzyme production by both free and immobilized cells of the yeast were temperature of 15 degrees C and initial pH of 8.0. The optimal incubation times were 72 and 96 h for immobilized and free cells, respectively. Immobilized cells were reused in 3 successive reaction cycles without any loss in the maximum protease activity. Little decreases in the protease activity were observed in 4 and 5 cycles. Under the optimized conditions, the maximum enzyme activities were determined as 12.1 and 13.5 U/mL for free and immobilized cells, respectively. This is a first attempt on cold-active alkaline protease production by free and/or immobilized cells of yeasts. Besides, the protease activity of the yeast C. victoriae CA-8 was investigated for the first time in the present study.
Sheep Wool Protein Hydrolysate: A New Peptone Source for Microorganisms
(Wiley, 2016) Taskin, Mesut; Unver, Yagmur; Firat, Abdulhadi; Ortucu, Serkan; Yildiz, Melike
BACKGROUND: Peptones are one of the most expensive components of microbial culture media. The present study was performed to produce microbial peptone from sheep wool using a new chemical process. RESULTS: Wool peptone (WP) was found to have high protein (67.8 g per 100 g) and ash (29.2 g per 100 g) contents. Glutamic acid was the most abundant amino acid inWP with a content of 8175 mg per 100 g). Wool peptone (WP) also had a high content (5042 mg per 100 g) of cystine, a sulphur-containing amino acid. Optimal concentration of WP was determined as 5 g L-1 for the fungi and 6 g L-1 for the bacteria. Staphylococcus aureus showed very poor growth performance in WP medium. Growth performances of Saccharomyces cerevisiae, Bacillus subtilis and Penicillium chrysogenum were at moderate levels in WP medium. The best growth performance for Aspergillus niger was observed in WP medium with a biomass production of 8.17 g L-1. Second best growth performance for Escherichia coli was achieved with WP among the tested peptones. CONCLUSION: Wool peptone (WP) was shown to be a good growth substrate, especially for A. niger and E. coli. This is the first investigation on use of wool as peptone source or substrate for microorganisms. (C) 2016 Society of Chemical Industry