Person: KARAMAN, ELİF
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ELİF
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KARAMAN
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Publication Open Access Large-Scale Production of Anti-RNase A VHH Expressed in pyrG Auxotrophic Aspergillus oryzae(2023-05-01) Karaman, Elif; Eyupoglu, Alp Ertunga; Mahmoudi Azar, Lena; Uysal, Serdar; KARAMAN, ELİF; UYSAL, SERDARNanobodies, also referred to as VHH antibodies, are the smallest fragments of naturally produced camelid antibodies and are ideal affinity reagents due to their remarkable properties. They are considered an alternative to monoclonal antibodies (mAbs) with potential utility in imaging, diagnostic, and other biotechnological applications given the difficulties associated with mAb expression.Aspergillus oryzae (A. oryzae)is a potential system for the large-scale expression and production of functional VHH antibodies that can be used to meet the demand for affinity reagents. In this study, anti-RNase A VHH was expressed under the control of the glucoamylase promoter inpyrGauxotrophicA. oryzaegrown in a fermenter. The feature ofpyrGauxotrophy, selected for the construction of a stable and efficient platform, was established using homologous recombination. Pull-down assay, size exclusion chromatography, and surface plasmon resonance were used to confirm the binding specificity of anti-RNase A VHH to RNase A. The affinity of anti-RNase A VHH was nearly 18.3-fold higher (1.9 nM) when expressed inpyrGauxotrophicA. oryzaerather than inEscherichia coli. This demonstrates thatpyrGauxotrophicA. oryzaeis a practical, industrially scalable, and promising biotechnological platform for the large-scale production of functional VHH antibodies with high binding activity.Publication Metadata only Reduction of methomyl and acetamiprid residues from tomatoes after various household washing solutions(2017-01-01T00:00:00Z) Rasolonjatovo, M. Angela; Cemek, Mustafa; CENGİZ, MELİKE; Ortac, Deniz; Konuk, H. Buesra; Karaman, ELİF; Kocaman, A. Tuba; Gones, Sadik; KARAMAN, ELİFThe removal of pesticide residues is essential in order to control and reduce the harmful effects of pesticides commonly used in agriculture. The aim of this study was to investigate the effectiveness of 18 different household washing solutions for the reduction of methomyl and acetamiprid residues from tomatoes. These basic household solutions were used to prepare washing solution to remove pesticides. During this study, tomatoes were divided into two groups: in the first group, tomatoes were treated with the pesticides and the second group was washed with household solutions (distilled water, acetic acid, sodium chloride, ethanol, sodium bicarbonate, hydrogen peroxide, sodium hypochlorite, and liquid soap, lemon juice, mineral water, zam-zam water, whey, milk, and their combinations) after treating with pesticides. The pesticides residues in all samples were extracted by the QuEChERS technique, analyzed by LC-MS/MS. The results showed significant reduction in residues of all washed groups compared with the control (p < 0.01). Acetic acid, sodium chloride+sodium bicarbonate, zam-zam water, acetic acid+sodium chloride, sodium hypochlorite, milk, acetic acid+sodium chloride+sodium bicarbonate, liquid whey, liquid soap, lemon juice, ethanol, and sodium bicarbonate were found to remove more than 50% of both pesticide residues. The effectiveness of washing solutions was different for every type of pesticide. In the same way, blend of washing solutions has shown a synergetic effect on the removal of pesticides and was more effective compared to one solution. This research has raised the potential use of household solutions easily found at home that could be used to remove pesticide residues.Publication Metadata only Investigating the Antioxidant Capacity of Lunasin Expressed in Aspergillus oryzae(2024-01-01) KARAMAN E.; Albayrak C.; UYSAL S.; KARAMAN, ELİF; ALBAYRAK, CEM; UYSAL, SERDARObjective: Lunasin is a bioactive protein that possesses anti-carcinogenic, anti-inflammatory, and antioxidant properties. Traditional isolation methods are resource-intensive, and chemical synthesis faces cost and environmental issues. This study aims to achieve cost-effective lunasin expression in Aspergillus oryzae with a focus on exploring its antioxidant properties in vitro. Materials and Methods: The expression vector carrying four lunasin sequences fused with amylase and an 8xHis-tag was introduced into pyrG auxotrophic A. oryzae. Subsequently, the recombinant protein was purified using metal affinity chromatography. The study uses sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analyses, and size-exclusion chromatography to evaluate the composition and purity of the protein, a linoleic acid assay to demonstrate the inhibitory effect on lipid peroxidation, and the 2,2\"-azinobis-[3-ethylbenzothiazoline-6-sulfonic acid] ABTS) assay to evaluate the radical scavenging activity. Results: SDS-PAGE and western blot analyses confirmed sustained lunasin expression in A. oryzae, appearing in both fusion and non-fusion forms. Yields were 5.8 mg/L for non-fusion and 4 mg/L for fusion lunasin expression. Moreover, 0.1 μM non-fusion lunasin surpassed α-tocopherol and butylated hydroxyanisole (BHA; p < 0.05) in reducing lipid peroxidation at 4 and 72 h. Unlike the fusion lunasin, the non-fusion lunasin displayed concentration- and time-independent inhibitory effects on linoleic acid peroxidation as well as significant ABTS scavenging activity (p < 0.05). Conclusion: The study has shown for the first time A. oryzae to efficiently express and secrete both fusion and non-fusion lunasin proteins in a soluble form, with the non-fusion lunasin exhibiting superior antioxidant effectiveness compared to the fusion lunasin. The findings underscore A. oryzae\"s potential as a promising host for producing functional lunasin with antioxidant properties, opening avenues for broader applications in biotechnology and bioactive peptides.