Browsing by Subject "Enzymes"

Now showing 1 - 5 of 5

Open Access
Biocatalytic protein membranes fabricated by electrospinning
(Elsevier B.V., 2016) Kabay, G.; Kaleli, G.; Sultanova, Z.; Ölmez, T. T.; Şeker, U. Ö. Ş.; Mutlu, M.
In this study, a protein-based catalytic membrane was produced by electrospinning. Membrane activity was characterised in terms of response current for various glucose concentrations. We focused on the preparation of a scaffold by converting a globular protein to other structural forms using catastrophic solvents. A scaffolding protein, bovine serum albumin, and an enzyme, glucose oxidase (GOD), were selected as a model natural carrier matrix and a biologically active agent, respectively. Beta-mercaptoethanol (β-ME) was used to convert the globular protein to an amyloid-like form. A structural stabilising agent, 2,2,2-triflouroethanol (TFE), was used to maintain the final α-helical structure of the amyloid-like protein. The TFE:PBS (phosphate-buffered saline) ratio and various electrospinning parameters were analysed to minimise activity loss. Using this approach, we applied electrospinning to an active enzyme to obtain biocatalytic nanofibrous membranes. After optimising the protein electrospinning process, the activities of the protein nanofibrous membranes were monitored. GOD remained active in the new membrane structure. The highest enzyme activity was observed for the membranes prepared with a 1.5:1 (v:v) TFE:PBS solvent ratio. In that particular case, the immobilized enzyme created a current of 0.7 μA and the apparent activity was 2547 ± 132 U/m2.
Open Access
Catalytic supramolecular self-assembled peptide nanostructures for ester hydrolysis
(Royal Society of Chemistry, 2016) Gulseren, G.; Khalily, M. A.; Tekinay, A. B.; Güler, Mustafa O.
Essential amino acids in catalytic sites of native enzymes are important in nature inspired catalyst designs. Active sites of enzymes contain the coordinated assembly of multiple amino acids, and catalytic action is generated by the dynamic interactions among multiple residues. However, catalysis studies are limited by the complex and dynamic structure of the enzyme; and it is difficult to exclusively attribute a given function to a specific residue. Minimalistic approaches involving artificial catalytic sites are promising for the investigation of the enzyme function in the absence of non-essential protein components, and self-assembling peptide nanostructures are especially advantageous in this context. Here we demonstrate the design and characterization of an enzyme-mimetic catalytic nanosystem presenting essential residues (Ser, His, Asp). The function of each residue and its combinations on the nanostructures in hydrolysis reaction was studied. The catalytic self-assembled nanostructures were used for efficient ester hydrolysis such as a model substrate (pNPA) and a natural substrate (acetylcholine) highlighting the key role of self-assembly in catalytic domain formation to test the efficiency of the de novo designed catalyst as a catalytic triad model.
Open Access
Cellular biocatalysts using synthetic genetic circuits for prolonged and durable enzymatic activity
(Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, 2019) Ahan, Recep Erdem; Şaltepe, Behide; Apaydın, Onur; Şeker, Urartu Özgür Şafak
Cellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole‐cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole‐cell biocatalysts. We employed two different strategies to increase the stability of whole‐cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole‐cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.
Open Access
Effect of oxygen supply on metabolism of immobilized and suspended Escherichia coli
(John Wiley & Sons Inc, New York, NY, United States, 1996) Inanç, E.; Miller J. E.; DiBiasio, D.
The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded β-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased β-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of β-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.The effect of reduced oxygen supply on the production of a recombinant protein (plasmid-encoded β-galactosidase) was investigated in Escherichia coli. A novel modified bubble tank reactor was used to provide a direct comparison between immobilized and suspended cells in identical environments except for the immobilization matrix. Decreased oxygen supply led to increased β-galactosidase synthesis by both immobilized and suspended cells. Immobilized cells produced similar amounts of β-galactosidase as the suspended cells. Lactose consumption and acetate production, on a per cell basis, were significantly higher in immobilized cells, suggesting that immobilized cells utilized fermentative metabolism. However, a transport analysis of the immobilized cell system showed that immobilized cells were not subject to either external or internal mass transfer gradients.
Open Access
In situ synthesis of biomolecule encapsulated gold-cross-linked poly(ethylene glycol) nanocomposite as biosensing platform: A model study
(Elsevier BV, 2010) Odaci, D.; Kahveci, M.U.; Sahkulubey, E.L.; Ozdemir, C.; Uyar, Tamer; Timur, S.; Yagci Y.
In situ synthesis of poly(ethylene glycol) (PEG) hydrogels containing gold nanoparticles(AuNPs) and glucose oxidase (GOx) enzyme by photo-induced electron transfer process was reported here and applied in electrochemical glucose biosensing as the model system. Newly designed bionanocomposite matrix by simple one-step fabrication offered a good contact between the active site of the enzyme and AuNPs inside the network that caused the promotion in the electron transfer properties that was evidenced by cyclic voltammetryas well as higher amperometric biosensing responses in comparing with response signals obtained from the matrix without AuNPs. As well as some parameters important in the optimization studies such as optimum pH, enzyme loading and AuNP amount, the analytical characteristics of the biosensor (AuNP/GOx) were examined by the monitoring of chronoamperometric response due to the oxygen consumption through the enzymatic reaction at − 0.7 V under optimized conditions at sodium acetate buffer (50 mM, pH 4.0) and the linear graph was obtained in the range of 0.1–1.0 mM glucose. The detection limit (LOD) of the biosensor was calculated as 0.06 mM by using the signal to noise ratio of 3. Moreover, the presence of AuNPs was visualized by TEM. Finally, the biosensor was applied for glucose analysis for some beverages and obtained data were compared with HPLC as the reference method to test the possible matrix effect due to the nature of the samples.