Enzymatic degradation of azo dyes methylene blue and congo red with peroxidase purified from cauliflower using affinity chromatography technique: Kinetic study, optimization and metal binding activity.

The effective results of the enzymatic decolorization of industrial azo dyes found in wastewater, which cause serious health and environmental problems, with peroxidases have recently increased the interest in these enzyme sources. Redox-mediated decolorization of Methylene Blue and Congo Red azo dyes with cauliflower (Brassica oleracea var. botrytis L.) peroxidase (CPOD) purified in one step using 4-amino 3-bromo 2-methyl benzohydrazide molecule was investigated for the first time. The inhibition effect of this molecule, which is used as a ligand in affinity chromatography, on the CPOD enzyme was investigated. The Ki and IC50 values for this enzyme were calculated as 0.113 ± 0.012 mM and 0.196 ± 0.011 mM, respectively. With the affinity gel obtained by binding to the Sepharose-4B-l-tyrosine matrix of this molecule, which shows a reversible inhibition effect, the purification values of CPOD enzyme were determined as 562-fold with a specific activity of 50,250 U mg-1. The purity of the enzyme was checked by the SDS-PAGE technique and its molecular weight was determined. A single band at 44 kDa was observed for the CPOD enzyme. In dye decolorization studies, the effects of dye, enzyme, and hydrogen peroxide concentrations as well as time, pH, and temperature were investigated. The profiles of the optimum conditions for both dyes were similar, and the percentages of decolorization of Methylene Blue and Congo Red under these conditions were 89% and 83%, respectively, at the end of the 40 min reaction time. Again, when examining the effect of metal ions on enzyme activity, it was found that there was no significant negative change in CPOD.

Investigation of biological activities of various 1,2,3-triazole compounds: Their effects on cholinesterase enzymes, determination of antioxidant capacity and antimicrobial activity.

1,2,3-triazoles are pharmaceutically significant compounds that have attracted recent interest from medicinal chemists because of their important biological activities. Addressed herein, some 1,2,3-triazoles were synthesized to investigate the inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes, antioxidant capacity, and antimicrobial effect. The antioxidant profile of 1,2,3-triazoles determined by varied bioanalytical antioxidant methods, including 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.+ ), 1,1-diphenyl-2-picrylhydrazil (DPPH·), cupric ion (Cu2+ ) and ferric ion (Fe3+ ) ascorbic acid, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) were used as the standard compounds. In addition, the antibacterial and antifungal activities of these compounds were investigated against seven bacteria and three fungal species using the hollow agar method. As a result of these studies, it was determined that compound 4 showed the best antimicrobial activity and antioxidant activity close to the standards. Inhibitory effects and kinetic studies of these molecules on cholinesterase enzymes were performed. According to the results obtained, compound 4 showed stronger AChE inhibition and compound 3 stronger BChE inhibition compared to other compounds. In kinetic studies, it was found that AChE showed noncompetitive inhibition by compound 4, and BChE showed competitive inhibition by compound 3.

A new approach for affinity-based purification of horseradish peroxidase.

We have developed efficient procedure for isolation of horseradish peroxidase (HRP) using aminobenzohydrazide-based affinity chromatography. Sepharose 4B-bounded aminobenzohydrazides are suitable for long-term use and large-scale purification. In this study, 26 aminobenzohydrazide derivatives were synthesized, characterized and defined as new HRP inhibitors. In addition, detailed inhibition effects of these molecules on HRP enzyme were investigated. Affinity matrix was formed by bonding aminobenzohydrazides, which exhibited inhibitory activity to sepharose-4B-l-tyrosine. HRP was isolated from crude homogenate in single step and purification factors were recorded as 1,151-fold (recovery of 8.5%) with 4-amino 3-bromo benzohydrazide and as 166.16-fold (recovery of 16.67 %) with 3-amino 4-chloro benzohydrazide.

Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique.

In this study, an effective single step affinity method is presented for purifying plant peroxidase (POD) enzymes from radish species. This method make possible to purify the enzymes in high yield and purity. Briefly, 10 different 4-amino benzohydrazide derivatives were synthesized and identified as new competitive POD inhibitors. Then, these derivatives were coupled to Sepharose 4B-L-Tyrosine support matrix by diazotization to form the affinity gels. Purification factors were recorded as 54.8% yield - 665-fold, 33.8% yield - 613-fold, 22.7% yield - 595-fold, 34.4% yield - 781-fold, 40.9% yield - 282-fold for turnip (T-POD), black radish (BR-POD), daikon (D-POD), sweet radish (SR-POD) and kohlrabi radish, (KR-POD), respectively. It has also been shown that the affinity gels, which prepared using the 4-amino 3-bromo benzohydrazide and 4-amino 2-nitro benzohydrazide molecules, capable to purify all radish species POD enzymes in high purity and yield.

Secondary Sulfonamides as Effective Lactoperoxidase Inhibitors.

Secondary sulfonamides (4a-8h) incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide, and trihydroxybenzamide and possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups were synthesized. Lactoperoxidase (LPO, E.C.1.11.1.7), as a natural antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives (4a-8h) were examined against LPO. The obtained results reveal that secondary sulfonamide derivatives (4a-8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives (4a-8h) were found in the range of 1.096 × 10-3 to 1203.83 µM against LPO. However, the most effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a), with Ki values of 1.096 × 10-3 ± 0.471 × 10-3 µM as non-competitive inhibition.

Biconically tapered fiber optic probes for rapid label-free immunoassays.

We report use of U-shaped biconically tapered optical fibers (BTOF) as probes for label-free immunoassays. The tapered regions of the sensors were functionalized by immobilization of immunoglobulin-G (Ig-G) and tested for detection of anti-IgG at concentrations of 50 ng/mL to 50 µg/mL. Antibody-antigen reaction creates a biological nanolayer modifying the waveguide structure leading to a change in the sensor signal, which allows real-time monitoring. The kinetics of the antibody (mouse Ig-G)-antigen (rabbit anti-mouse IgG) reactions was studied. Hydrofluoric acid treatment makes the sensitive region thinner to enhance sensitivity, which we confirmed by experiments and simulations. The limit of detection for the sensor was estimated to be less than 50 ng/mL. Utilization of the rate of the sensor peak shift within the first few minutes of the antibody-antigen reaction is proposed as a rapid protein detection method.