Browsing by Author "Alpdagtas, Saadet"

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The Challenges of Using Nad+-Dependent Formate Dehydrogenases for Co2 Conversion
(Taylor & Francis Ltd, 2022) Alpdagtas, Saadet; Turunen, Ossi; Valjakka, Jarkko; Binay, Baris
In recent years, CO2 reduction and utilization have been proposed as an innovative solution for global warming and the ever-growing energy and raw material demands. In contrast to various classical methods, including chemical, electrochemical, and photochemical methods, enzymatic methods offer a green and sustainable option for CO2 conversion. In addition, enzymatic hydrogenation of CO2 into platform chemicals could be used to produce economically useful hydrogen storage materials, making it a win-win strategy. The thermodynamic and kinetic stability of the CO2 molecule makes its utilization a challenging task. However, Nicotine adenine dinucleotide (NAD(+))-dependent formate dehydrogenases (FDHs), which have high selectivity and specificity, are attractive catalysts to overcome this issue and convert CO2 into fuels and renewable chemicals. It is necessary to improve the stability, cofactor necessity, and CO2 conversion efficiency of these enzymes, such as by combining them with appropriate hybrid systems. However, metal-independent, NAD(+)-dependent FDHs, and their CO2 reduction activity have received limited attention to date. This review outlines the CO2 reduction ability of these enzymes as well as their properties, reaction mechanisms, immobilization strategies, and integration with electrochemical and photochemical systems for the production of formic acid or formate. The biotechnological applications of FDH, future perspectives, barriers to CO2 reduction with FDH, and aspects that must be further developed are briefly summarized. We propose that constructing hybrid systems that include NAD(+)-dependent FDHs is a promising approach to convert CO2 and strengthen the sustainable carbon bio-economy.
Discovery of an Acidic, Thermostable and Highly Nadp+ Dependent Formate Dehydrogenase From Lactobacillus Buchneri Nrrl B-30929
(Springer, 2018) Alpdagtas, Saadet; Yucel, Sevil; Kapkac, Handan Acelya; Liu, Siqing; Binay, Baris
To identify a robust NADP(+) dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties. A new NADP(+) dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6 x histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8-6.2). Its optimum temperature was 60 A degrees C and 50 A degrees C with NADP(+) and NAD(+), respectively and its T-m value was 78 A degrees C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K-M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP(+)), NADP(+) and NAD(+), respectively. An NADP(+) dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.
Dmso Tolerant Nad(P)h Recycler Enzyme From a Pathogenic Bacterium, Burkholderia Dolosa Pc543: Effect of N-/C-terminal His Tag Extension on Protein Solubility and Activity
(Wiley, 2018) Alpdagtas, Saadet; Celik, Ayhan; Ertan, Fatma; Binay, Baris
NAD(P)(+) dependent formate dehydrogenase (FDH) is an oxidoreductase used as a biocatalyst to regenerate NAD(P)H in reductase-mediated chiral synthesis reactions. Solvent stability and the need to reduce NADP(+) to NADPH, due to the high cost of NADPH, are required features in the industrial usage of FDHs. Therefore, we aimed to identify a novel, robust NADP(+) dependent FDH and evaluate the effect of N- and C- terminus His tag extensions on protein solubility and activity. Herein, we report a novel, DMSO tolerant formate dehydrogenase (BdFDH), which has dual coenzyme specificity and tolerance to acidic pH, from Burkholderia dolosa PC543. N- and C-terminus His-tagged BdFDHs were expressed separately in Escherichia coli BL21 (DE3). The C-terminal His-tagged BdFDH was soluble and active whereas the N-terminal version was not. The enzyme displays dual coenzyme specificity and resistance to some organic solvents, particularly DMSO, and is able to tolerate acidic pH conditions. The apparent K-M values for NADP(+), NAD(+) and sodium formate (with NADP(+)), are 1.17, 14.7 and 5.66 mM, respectively. As a result, due to its DMSO tolerance and coenzyme preference, this enzyme can be utilized as an NAD(P)H recycler in several biotransformations particularly when carried out under acidic conditions. Moreover, it can be said that the position of the His tag extension may affect the enzyme solubility and functionality.
Evaluation of Current Diagnostic Methods for Covid-19
(Aip Publishing, 2020) Alpdagtas, Saadet; Ilhan, Elif; Uysal, Ebru; Sengor, Mustafa; Ustundag, Cem Bulent; Gunduz, Oguzhan
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent responsible for the coronavirus disease of 2019 (COVID-19), which triggers lung failure, pneumonia, and multi-organ dysfunction. This enveloped, positive sense and single-stranded RNA virus can be transmitted through aerosol droplets, direct and indirect contacts. Thus, SARS-CoV-2 is highly contagious and has reached a pandemic level in a few months. Since COVID-19 has caused numerous human casualties and severe economic loss posing a global threat, the development of readily available, accurate, fast, and cost-effective diagnostic techniques in hospitals and in any places where humans spread the virus is urgently required. COVID-19 can be diagnosed by clinical findings and several laboratory tests. These tests may include virus isolation, nucleic acid-based molecular assays like real-time polymerase chain reactions, antigen or antibody-based immunological assays such as rapid immunochromatographic tests, enzyme-linked immunosorbent assays, immunofluorescence techniques, and indirect fluorescent antibody techniques, electrochemical sensors, etc. However, current methods should be developed by novel approaches for sensitive, specific, and accurate diagnosis of COVID-19 cases to control and prevent this outbreak. Thus, this review will cover an overview and comparison of multiple reports and commercially available kits that include molecular tests, immunoassays, and sensor-based diagnostic methods for diagnosis of COVID-19. The pros and cons of these methods and future perspectives will be thoroughly evaluated and discussed.
Functional Role of Crosslinking in Alginate Scaffold for Drug Delivery and Tissue Engineering: a Review
(Pergamon-elsevier Science Ltd, 2021) Aguero, Lisette; Alpdagtas, Saadet; Ilhan, Elif; Zaldivar-Silva, Dionisio; Gunduz, Oguzhan
The actual tendency exhibits continuous increment to use alginate polysaccharides in drug delivery and tissue engineering fields due to its attractive properties. Alginate pH-sensitiveness and its fabrication techniques have been extensively reported in the literature. However, in this review, the essential role of crosslinking process in the scaffold design will be highlighted. We discuss the possibility to use a different type of crosslinking to create proper biomimetic alginate hydrogels, emphasizing the possibility to improve alginate mechanical properties through chemical modification by covalent crosslinking or ionic combination with other polymeric materials. At the same time, the relevance of the degradation process and biocompatibility in these applications is also detailed described.
Identification of Redox Activators for Continuous Reactivation of Glyoxal Oxidase From Trametes Versicolor in a Two-Enzyme Reaction Cascade
(Nature Portfolio, 2024) Alpdagtas, Saadet; Jankowski, Nina; Urlacher, Vlada B.; Koschorreck, Katja
Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O-2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2 '-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.
Nadp+-Dependent Formate Dehydrogenase: a Review
(Taylor & Francis Ltd, 2021) Alpdagtas, Saadet; Binay, Baris
NADPH-dependent oxidoreductases are crucial biocatalysts for the industrial production of chiral compounds. For in situ recycling of required expensive cofactors in this biosynthetic process, NAD(P)(+)-dependent formate dehydrogenases (FDHs) are wanted to be employed as redox biocatalysts due to their greener and process friendly nature. However, their utilization is limited by their undesired cofactor preference that strongly prone to NAD(+) more than NADP(+) and catalytic efficiency. To mine NADP(+)-dependent FDHs in nature by the guidance of bioinformatic tools or re-engineering of their NAD(+)-dependent equivalents to get an applicable recycler are attractive topics in bioengineering. It can be said that, up to now, the attempts to switch the cofactor preference of the FDHs generally have resulted in NADP(+)-dependent enzymes that have not to catch the desired catalytic efficiencies or stability. In this review, all studies about the native NADP(+)-dependent FDHs and also engineered equivalents that reconstructed with different protein engineering approaches for altering the coenzyme specificity are outlined. To switch the coenzyme preference of FDHs or to find the native NADP(+)-dependent FDHs will be the hot topics in bioengineering until finding a feasible regenerator. Therefore, this study will be a useful guide to get a pathway for designing or discovering novel NADP(+)-dependent FDHs.
Tailoring of Recombinant Fdh: Effect of Histidine Tag Location on Solubility and Catalytic Properties of Chaetomium Thermophilum Formate Dehydrogenase (Ctfdh)
(Taylor & Francis inc, 2019) Esen, Hacer; Alpdagtas, Saadet; Cakar, Mehmet Mervan; Binay, Baris
Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD(+) dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD(+) to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.