Ayhan Çelik

The olive mill wastewater as substrate for single cell oil production by Zygomycetes.

The conversion of olive mill wastewater (OMW) into high added value lipids containing polyunsaturated fatty acids (PUFA), in parallel with a significant phenolic removal by selected strains of Zygomycetes, is reported here for the first time. The growth of Mortierella isabellina, Mortierella ramanniana, Cunninghamella echinulata, Mucor sp., Thamnidium elegans and Zygorhynchus moelleri on solidified media was not significantly affected by the presence of OMW used in the growth medium up to 50% (v/v). Kinetic parameter values and conversion yields, estimated using a mathematical model which was fitted on the experimental data originated from submerged cultures, shows the ability of some Zygomycetes (i.e. T. elegans and Z. moelleri) to grow on OMW and accumulate storage material, i.e. lipids rich in PUFA, and these findings open new perspectives in OMW management and valorization. In liquid media containing OMW as sole carbon source, T. elegans and Z. moelleri produced 4.4 and 3.5g/L cell mass in surface (SC) and submerged (SMC) cultures, respectively, containing around 60% (w/w) of lipids. Oleic and palmitic acids were the predominant fatty acids. Gamma-linolenic acid was found in high percentages (up to 17.7%, w/w) in the lipid of Z. moelleri, in SMC with OMW as sole carbon source, while PUFA biosynthesis was not favored in SC.

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co(2+) modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD(+) with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.

An unusually cold active nitroreductase for prodrug activations

A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.

Highly stable and reusable immobilized formate dehydrogenases: Promising biocatalysts for in situ regeneration of NADH

Summary This study aimed to prepare robust immobilized formate dehydrogenase (FDH) preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150), Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU), and Immobead 150 support functionalized with aldehyde groups (FDHIALD). The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t 1/2) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.

Biological evaluation and molecular docking studies of nitro benzamide derivatives with respect to in vitro anti-inflammatory activity

&NA; A series of nitro substituted benzamide derivatives were synthesized and evaluated for their potential anti‐inflammatory activities in vitro. Firstly, all compounds (1–6) were screened for their inhibitory capacity on LPS induced nitric oxide (NO) production in RAW264.7 macrophages. Compounds 5 and 6 demonstrated significantly high inhibition capacities in a dose‐dependent manner with IC50 values of 3.7 and 5.3 &mgr;M, respectively. These two compounds were also accompanied by no cytotoxicity at the studied concentrations (max 50 &mgr;M) in macrophages. Molecular docking analysis on iNOS revealed that compounds 5 and 6 bind to the enzyme more efficiently compared to other compounds due to having optimum number of nitro groups, orientations and polarizabilities. In addition, 5 and 6 demonstrated distinct regulatory mechanisms for the expression of the iNOS enzyme at the mRNA and protein levels. Specifically, both suppressed expressions of COX‐2, IL‐1&bgr; and TNF‐&agr; significantly, at 10 and 20 &mgr;M. However, only compound 6 significantly and considerably decreased LPS‐induced secretion of IL‐1&bgr; and TNF‐&agr;. These results suggest that compound 6 may be a multi‐potent promising lead compound for further optimization in structure and as well as for in vivo validation studies. Graphical abstract: Figure. No caption available. HighlightsSix (1‐6) nitro substituted benzamide derivatives were synthesized.Their anti‐inflammatory activities were screened by nitric oxide inhibition assay in LPS induced RAW264.7 macrophages.Two lead compounds were identified and investigated for NO inhibitory IC50 values and dose‐response relations.Molecular docking studies on iNOS enzyme supported the biological data.The effects of lead compounds on iNOS, COX‐2, IL‐1&bgr; and TNF‐&agr; expression were investigated.

Antiproliferative activity of Humulus lupulus extracts on human hepatoma (Hep3B), colon (HT-29) cancer cells and proteases, tyrosinase, β-lactamase enzyme inhibition studies

Abstract The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72 h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6–1 mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1 mg/mL for 72 h in Hep3B cells and 0.1–0.2 mg/mL for 48 and 72 h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (Vmax) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and β-lactamase (penicillinase).

Prodrugs For Nitroreductase Based Cancer Therapy- 1 Metabolite Profile, Cell Cytotoxicity and Molecular Modeling Interactions of Nitro Benzamides with Ssap-NtrB

BACKGROUND Directed Enzyme Prodrugs Therapy (DEPT) as an alternative method against conventional cancer treatments, in which the non-toxic prodrugs is converted to highly cytotoxic derivative, has attracted an ample attention in recent years for cancer therapy studies. OBJECTIVE The metabolite profile, cell cytotoxicity and molecular modeling interactions of a series of nitro benzamides with Ssap-NtrB were investigated in this study. METHOD A series of nitro-substituted benzamide prodrugs (1-4) were synthesized and firstly investigated their enzymatic reduction by Ssap-NtrB (S. saprophyticus Nitroreductase B) using HPLC analysis. Resulting metabolites were analyzed by LC-MS/MS. Molecular docking studies were performed with the aim of investigating the relationship between nitro benzamide structures (prodrugs 1-4) and Ssap-NtrB at the molecular level. Cell viability assay was conducted on two cancer cell lines, hepatoma (Hep3B) and colon (HT-29) cancer models and healthy cell model HUVEC. Upon reduction of benzamide prodrugs by Ssap-NtrB, the corresponding amine effectors were tested in a cell line panel comprising PC-3, Hep3B and HUVEC cells and were compared with the established NTR substrates, CB1954 (an aziridinyl dinitrobenzamide). RESULTS Cell viability assay resulted in while prodrugs 1, 2 and 3 had no remarkable cytotoxic effects, prodrug 4 showed the differential effect, showing moderate cytotoxicity with Hep3B and HUVEC. The metabolites that obtained from the reduction of nitro benzamide prodrugs (1-4) by Ssap-NtrB, showed differential cytotoxic effects, with none toxic for HUVEC cells, moderate toxic for Hep3B cells, but highly toxic for PC3 cells. CONCLUSION Amongst all metabolites of prodrugs after Ssap-NtrB reduction, N-(2,4- dinitrophenyl)-4-nitrobenzamide (3) was efficient and toxic in PC3 cells as comparable as CB1954. Kinetic parameters, molecular docking and HPLC results also confirm that prodrug 3 is better for Ssap-NtrB than 1, 2 and 4 or known cancer prodrugs of CB1954 and SN23862, demonstrating that prodrug 3 is an efficient candidate for NTR based cancer therapy.

A new lipase as a pharmaceutical target for battling infections caused by Staphylococcus aureus: Gene cloning and biochemical characterization

Staphylococcus aureus lipases along with other cell‐wall‐associated proteins and enzymes (i.e., catalase, coagulase, protease, hyaluronidase, and β‐lactamase) play important roles in the pathogenesis of S. aureus and are important subject of drug targeting. The appearance of antibiotic‐resistant types of pathogenic S. aureus (e.g., methicillin‐resistant S. aureus, MRSA) is a worldwide medical problem. In the present work, a novel lipase from a newly isolated MRSA strain from a cow with subclinical mastitis was cloned and biochemically characterized. The mature part of the lipase was expressed in Escherichia coli and purified by nickel affinity chromatography. It displays a high lipase activity at pH 8.0 and 25 °C using p‐nitrophenyl palmitate and has a preference for medium–long‐chain substrates of p‐nitrophenyl esters (pNPC10–C16). Furthermore, in search of inhibitors, the effect of farnesol on the growth of S. aureus and the lipase activity was also studied. Farnesol inhibits the growth of S. aureus and is a mixed‐type inhibitor with Ki and Ki′ values of 0.2 and 1.2 mmol L−1, respectively. A lipase with known properties could not only serve as a template for developing inhibitors for S. aureus but also a valuable addition to enzyme toolbox of biocatalysis. The discovery of this lipase can be potentially important and could provide a new target for pharmaceutical intervention against S. aureus infection.

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

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 KM 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.

Modification of existing antibiotics in the form of precursor prodrugs that can be subsequently activated by nitroreductases of the target pathogen

The use of existing antibiotics in the form of prodrug followed by activation using enzymes of pathogenic origin could be a useful approach for antimicrobial therapy. To investigate this idea, a common antibiotic, sulfamethoxazole has been redesigned in the form of a prodrug by simple functional group replacement. Upon reductive activation by a type I nitroreductase from a pathogen, the drug displayed enhanced antimicrobial capacity. This strategy could improve the efficacy and selectively of antibiotics and reduce the incidence of resistance.