Photometric Assay Research Articles

Can splitting the blood sample for photometric chemical assay and immunoassay reduce turnaround time for chemical assay?

Can splitting the blood sample for photometric chemical assay and immunoassay reduce turnaround time for chemical assay?

Exploring the potential use of Caenorhabditis elegans as an animal model for evaluating chemical-induced intestinal dysfunction

Evaluating intestinal toxicity is crucial for identifying and preventing the harmful effects of environmental chemicals. Owing to the limitations of existing models in evaluating intestinal toxicity, the development of alternative models is urgently needed. This study explored the potential use of the nematode Caenorhabditis elegans as a model animal for assessing chemical-induced intestinal dysfunction. Changes in intestinal permeability and nutrient absorption in C. elegans individuals exposed to four intestine-disrupting chemicals (sodium dodecyl sulfate (SDS), dextran sulfate sodium (DSS), lipopolysaccharide (LPS) and ethanol) were examined using dye stain assays, an enzymatic photometric assay, and fluorescent probe uptake assays. Additionally, epigallocatechin-3-gallate (EGCG), an intestine-protecting phytochemical, was chosen to prevent ethanol-induced intestinal damage. The results indicated that SDS, DSS, LPS, and ethanol compromised the intestinal barrier in C. elegans. SDS had no effect on glucose absorption, but LPS, DSS, and ethanol inhibited or tended to inhibit glucose absorption. SDS, DSS, LPS, and ethanol reduced fatty acid absorption. LPS increased peptide absorption at a low dose but decreased it at a high dose; SDS, DSS, and ethanol attenuated peptide absorption. EGCG protected against the disruption of the intestinal barrier that was induced by ethanol treatment. These results suggest that C. elegans is a suitable surrogate model animal for evaluating chemical-induced intestinal dysfunction. These findings also provide new insights into the effects of SDS, DSS, LPS, and ethanol on intestinal function and highlight the potential of EGCG as a natural dietary intervention to protect individuals who use excess alcohol from intestinal injury.

Reductive dehalogenase of Dehalococcoides mccartyi strain CBDB1 reduces cobalt- containing metal complexes enabling anodic respiration.

Microorganisms capable of direct or mediated extracellular electron transfer (EET) have garnered significant attention for their various biotechnological applications, such as bioremediation, metal recovery, wastewater treatment, energy generation in microbial fuel cells, and microbial or enzymatic electrosynthesis. One microorganism of particular interest is the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1, known for its ability to reductively dehalogenate toxic and persistent halogenated organic compounds through organohalide respiration (OHR), using halogenated organics as terminal electron acceptors. A membrane-bound OHR protein complex couples electron transfer to proton translocation across the membrane, generating a proton motive force, which enables metabolism and proliferation. In this study we show that the halogenated compounds can be replaced with redox mediators that can putatively shuttle electrons between the OHR complex and the anode, coupling D. mccartyi cells to an electrode via mediated EET. We identified cobalt-containing metal complexes, referred to as cobalt chelates, as promising mediators using a photometric high throughput methyl viologen-based enzyme activity assay. Through various biochemical approaches, we show that cobalt chelates are specifically reduced by CBDB1 cells, putatively by the reductive dehalogenase subunit (RdhA) of the OHR complex. Using cyclic voltammetry, we also demonstrate that cobalt chelates exchange electrons with a gold electrode, making them promising candidates for bioelectrochemical cultivation. Furthermore, using the AlphaFold 2-calculated RdhA structure and molecular docking, we found that one of the identified cobalt chelates exhibits favorable binding to RdhA, with a binding energy of approximately -28 kJ mol-1. Taken together, our results indicate that bioelectrochemical cultivation of D. mccartyi with cobalt chelates as anode mediators, instead of toxic halogenated compounds, is feasible, which opens new perspectives for bioremediation and other biotechnological applications of strain CBDB1.

Facile photometric quantification of aldehyde content in oxidized starches using 2-aminobenzamide oxime as an aldehyde selective reagent

AbstractThe oxidation of starch to aldehyde starch or dialdehyde starch introduces aldehyde groups to the starch core structure. This functionalization enables a wide range of further derivatization, e.g., oxidation to the corresponding carboxylic acid or functionalization to the corresponding amine via imine formation and reduction, which can be used for cross-linking. Herein, we report a novel method for efficiently quantifying these aldehyde groups based on the selective adduct formation of 2-aminobenzamide oxime (ABAO) and aldehydes. We initially implemented this photometric assay for C6-oxidized aldehyde starch using a plate reader, enabling parallel analysis of multiple samples in a fraction of the time and consistent with the conventional titration methods. Complementing this stream-lined analytical approach, we present an abbreviated workflow for faster downstream processing of reaction samples toward analysis. All of this was performed at a very small scale, cutting costs for (expensive) reagents and solvents, as well as drastically reducing working time. Finally, we also demonstrate our assay's applicability for quantifying the two aldehyde groups formed in dialdehyde starch derived from NaIO4 oxidation. Graphical abstract

Bimetallic nanocolloidal plasmonic array for polyphenol characterization and calibration-free antioxidant capacity evaluation.

Abimetallic plasmonic nanoparticles-based approach for the untargeted evaluation of phenolic compounds (PC)-pattern and antioxidant capacity (AoC) is proposed. The rationale relies on the PC's ability to drive the formation of bimetallic silver/gold nanocolloidal 'probes' with different conformations. Ag/Au bimetallic nanostructures, according to the PCs' amount and class, return characteristic plasmonic and colorimetric tags. Plasmonic indexes are proposed to assess the dominant PC classes, while the colorimetric response, analyzed simply by a smartphone, is employed to obtain an AoC score, withoutcalibration. The methods were tested with PCs belonging to different chemical classes, and challenged to classify different food samples.The proposed approach allows PC-dominant class identification and AoC-evaluation consistent with HPLC-MS/MS and conventional photometric assays.

Evaluation of 4-fluoro-7-nitrobenzofurazan as a dual-function chromogenic and fluorogenic probe for tulathromycin and its innovative utility for development of two eco-friendly and high-through microwell assays for analysis of pharmaceutical formulations

Tulathromycin is a triamilide antibacterial drug which has been approved for use in the European Union and the United States for the treatment and prevention of bovine respiratory diseases. The aim of this study was the development of two innovative microwell spectrometric (photometric and fluorometric) assays for determination of tulathromycin in its pharmaceutical formulations. To achieve this goal, 4-fluoro-7-nitrobenzofurazan was investigated as a dual-function chromogenic and fluorogenic probe for tulathromycin. The reaction between tulathromycin and 4-fluoro-7-nitrobenzofurazan proceeded smoothly in an alkaline medium, resulting in the formation of a colored and fluorescent product. The product displayed a maximum light absorption at 475 nm and emitted fluorescence at 545 nm when excited at 475 nm. Extensive investigations were conducted to optimize the factors affecting the tulathromycin /4-fluoro-7-nitrobenzofurazan reaction, and the optimal conditions were established. Using these optimized conditions, both microwell-based photometric and fluorometric assays were developed. The calibration curves relating the absorbance and fluorescence intensities of the reaction product with the corresponding tulathromycin concentrations were generated. The absorbance-concentration relationship was found to be linear within a tulathromycin concentration range of 10–400 µg mL−1, with a limit of quantitation of 6.2 µg mL−1. On the other hand, the fluorescence-concentration relationship was linear within a concentration range of 0.04–1.2 µg mL−1, with a limit of quantitation of 0.06 µg mL−1. Rigorous validations of both assays’ procedures were performed, and both assays were successfully employed for the analysis of tulathromycin–containing pharmaceutical formulations (injections) with satisfactory accuracy and precision. The ecologically friendly assessment of both assays demonstrated their compliance with the principles of green analytical chemistry approaches. Moreover, the proposed microwell-based assays enabled the simultaneous analysis of multiple samples using small volumes, enabling high-throughput analysis. In conclusion, this study represents the first evaluation of 4-fluoro-7-nitrobenzofurazan as a probe with dual functionality for the microwell-based photometric and fluorometric analysis of tulathromycin. The developed assays serve as valuable analytical tools for ensuring the quality of tulathromycin ’s pharmaceutical formulations.

In Vitro Corrosion of Polyester-Coated Magnesium Alloy under pH-Static Conditions.

The resorption rate of bioresorbable implants requires tuning to match the desired field of application. The use of Mg as implant material is highly advantageous, as it provides sufficient mechanical strength combined with its biodegradability. Consequently, the implant vanishes after it has served its intended purpose, allowing the complete restoration of natural tissue and organ function. However, a biodegradable Mg implant requires a biodegradable coating to slow the rate of Mg corrosion, as a permanent coating would negate the benefits of using Mg as an implant material. Therefore, degradable polymers are the materials of choice, especially polyester-based coatings, such as PLLA, as they have been proven in clinical practice over the long term. Within this work, the degradation retarding effect of a physical barrier in form of four clinically relevant polyester-based coatings, poly-l-lactide (PLLA), poly-l-lactide-co-glycolide (PLGA), poly(l-lactide-co-PEG) triblock copolymer (PLLA-co-PEG), and polydioxanone (PDO), is investigated in vitro under pH-static conditions using CO2 gas to compensate pH changes due to Mg corrosion. Coating thicknesses of 7.5 to 8.3 μm were comparable to commercially available stent systems. Quantitative analysis of magnesium concentration in buffered test medium by a photometric assay allows real-time monitoring. Shielding effect of different polyesters through polymer coating and formation of a protective passivation layer beneath the polymer coating was observed and characterized using SEM and EDX techniques. Our finding was that even imperfect polymer layers provide a considerable protective effect, and the used in vitro setup matches reported in vivo observations regarding elemental composition of corrosion products.

Challenges in the determination of reactive oxygen species evolving during membrane water electrolysis for in situ ozone production

The treatment of ultrapure water with electrochemically produced O3 is a common means for disinfection yet leads to the formation of a variety of reactive oxygen species (ROS). The present study draws a comprehensive comparison between three commonly used photometric and fluorometric assays for ROS analysis and quantifies the individual signal responses for dissolved O3, ·OH and H2O2, respectively, to account for cross-sensitivities. By calibrating all combinations of assays and analytes, we developed a quantification procedure to reliably determine the actual ROS composition in ultrapure water environments for different operation conditions of a membrane water electrolyzer with PbO2 anodes down to concentrations of 0.97 μg L−1. While the ·OH formation rate can be described linearly over the observed current density range, substantial O3 evolution is only found for current densities of 0.75 A cm−2 and above (up to 3.7 μmol h−1 for J = 1.25 A cm−2). The formation of H2O2 is only observed when an organic carbon source is introduced into the solution. We further quantify the interference of H2O2 with the reading of the oxidation-reduction potential as a common water parameter and elaborate on its validity to monitor the peroxone process when both H2O2 and O3 are present simultaneously.

Open Meter Duo: Low-Cost Instrument for Fluorimetric Determination of Cholinesterase Activity.

Environmental screening is essential due to the increased occurrence of harmful substances in the environment. Open Meter Duo (OMD) is an open-source field photo/fluorimeter that uses an RGB diode that imitates a color according to the selected wavelength and uses a UV LED from the security kit diode as an excitation light source. The prepared PCB shield with a 3D-printed aperture was connected to Arduino UNO R4 WiFi. This system was used for the fluorescent detection of cholinesterase activity with the indoxyl acetate method. Carbofuran-a toxic pesticide-and donepezil-a drug used to treat Alzheimer's disease-were tested as model inhibitors of cholinesterase activity. The limit of detection of indoxyl acetate was 11.6 μmol/L, and the IC50 values of the inhibitors were evaluated. This system is optimized for wireless use in field analysis with added cloud support and power source. The time of analysis was 5 min for the fluorimetric assay and 20 min for the optional photometric assay. The time of field operation was approximately 4 h of continuous measurement. This system is ready to be used as a cheap and easy control platform for portable use in drug control and point-of-care testing.

Specific GAG ratios in the diagnosis of mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) screening is tedious and still performed by analysis of total glycosaminoglycans (GAG) using 1,9-dimethylmethylene blue (DMB) photometric assay, although false positive and negative tests have been reported. Analysis of differentiated GAGs have been pursued classically by gel electrophoresis or more recently by quantitative LC-MS assays. Secondary elevations of GAGs have been reported in urinary tract infections (UTI). In this manuscript, we describe the diagnostic accuracy of urinary GAG measurements by LC-MS for MPS typing in 68 untreated MPS and mucolipidosis (ML) patients, 183 controls and 153 UTI samples. We report age-dependent reference values and cut-offs for chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS) and specific GAG ratios. The use of HS/DS ratio in combination to GAG concentrations normalized to creatinine improves the diagnostic accuracy in MPS type I, II, VI and VII. In total 15 samples classified to the wrong MPS type could be correctly assigned using HS/DS ratio. Increased KS/HS ratio in addition to increased KS improves discrimination of MPS type IV by excluding false positives. Some samples of UTI patients showed elevation of specific GAGs, mainly CS, KS and KS/HS ratio and could be misclassified as MPS type IV. Finally, DMB photometric assay performed in MPS and ML samples reveal four false negative tests (sensitivity of 94%). In conclusion, specific GAG ratios in complement to quantitative GAG values obtained by LC-MS enhance discrimination of MPS types. Exclusion of patients with UTI improve diagnostic accuracy in MPS IV but not in other types.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs.Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10–200 μM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

In Brassica plants, glucosinolates are a diverse class of natural products, of which aliphatic methionine-derived glucosinolates are the most abundant form. Their structural diversity comes from the elongation of some side-chains by up to 9 carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Methylthioalkylmalate synthase (MAMS) catalyzes the iterative elongation process for aliphatic methionine-derived glucosinolates. Most biochemical studies on MAMS have been performed using liquid chromatography/mass spectrometry (LC/MS)-based assays or high-performance liquid chromatography (HPLC)-based assays. The LC/MS- and HPLC-based methods are endpoint assays, which cannot be monitored in real time and require a laborious process for data collection. These analytical methods are inefficient for performing multiple enzymatic assays needed to determine steady-state kinetic parameters or for mechanistic evaluation of pH-dependence and kinetic isotope effect studies. Although the function of MAMS has long been defined, there is a gap in knowledge as it pertains to biochemical characterization of this plant enzyme. Part of this may be due to the lack of efficient methods that can be used for this type of research. This chapter describes a continuous photometric assay to track MAMS activity in real time using the 4-aldrithiol reagent for reaction detection.

A dual-function chromogenic and fluorogenic benzofurazan probe for plazomicin and its innovative utility for development of two microwell assays with high throughput for analysis of drug substance and pharmaceutical formulations.

Plazomicin (PLZ) is a novel aminoglycoside which has been recently approved by The US Food and Drug Administration for the treatment of complicated urinary tract infections including acute pyelonephritis, caused by certain Enterobacteriaceae, in adult patients with limited or no options for alternative treatment. This study focuses on the development of microwell-based photometric and fluorometric assays for the quantitative determination of PLZ in its bulk drug substance and commercial pharmaceutical formulations (Zemedri® injections). Both assays utilize the dual-function chromogenic and fluorogenic properties of the 4-fluoro-7-nitrobenzofurazan (NBD-F) probe. The reaction between PLZ and NBD-F, conducted in a borate buffer at pH 8, resulted in the formation of a colored and fluorescent reaction product. The product exhibited maximum light absorption at 473 nm and emitted fluorescence at 541 nm when excited at 473 nm. Factors influencing the reaction between PLZ and NBD-F were thoroughly investigated, and optimal conditions were determined. Under the optimized reaction conditions, calibration curves were generated to establish the relationship between absorbance and fluorescence intensities of the reaction product with the corresponding PLZ concentrations. The absorbance-concentration relation was linear in a PLZ concentration range of 20-800 μg mL-1 with a limit of quantitation of 25 μg mL-1, while the fluorescence-concentration relation was linear in the concentration range of 0.05-1.5 μg mL-1 with a limit of quantitation of 0.08 μg mL-1. Both assays underwent validation and were successfully applied to the quantitation of PLZ in its bulk drug substance and pharmaceutical formulations (injections) with satisfactory accuracy and precision. The eco-friendliness/greenness assessment of the assays demonstrated that both assays comply with the requirements of green analytical chemistry approaches. Furthermore, the proposed microwell assay plates allowed for the simultaneous handling of numerous samples with micro-volumes, enabling high-throughput analysis. In conclusion, this study represents the first evaluation of NBD-F as a dual-function probe for the microwell-based photometric and fluorometric determination of PLZ. The developed assays serve as valuable analytical tools for the quality control of PLZ's bulk drug substance and pharmaceutical formulations.

Diagnostic Value of CRP, H-FABP, PCT, Lp-PLA2 and Cytokines in Stable Angina

Coronary artery disease (CAD) is a significant cause of worldwide mortality and morbidity. This study aims to evaluate the levels of serum H-FABP (fatty acid-binding protein), hs-CRP (high sensitivity- CPR), Lp-PLA2 (lipoprotein PLA2), PCT (procalcitonin) and cytokines, in addition to routinely used diagnostic tests, Troponin I (Trop I), Myoglobin (MYO) and Creatine kinase MB (CK-MB) in patients with stable angina to determine their sensitivity in diagnosing stable angina and facilitating faster decision-making in the emergency unit. The current study was performed on 86 patients complaining stable angina, at Nasiriyah Heart Center from October 2021 to October 2022. Eighty-six, healthy subjects (age-matched) were taken as a control group. Blood samples were collected in the emergency department. Serum levels of hs CRP, H-FABP, CK-MB, Trop I, MYO, Lp-PLA2 and PCT were determined using electro-chemiluminescence immunoassay. Blood sugar and serum total cholesterol, triglycerides, LDL, VLDL and HDL were determined using Cobas C311 photometric assays. Serum IL-6 was determined by using electro-chemiluminescence immunoassay, while, IL-9, IL-1β and TNF-α were assayed by ELISA. The study showed that the level of troponin I didn’t significantly change in patients with stable angina. However, compared with healthy controls, patients showed a significant increase in serum levels of CK-MB, myoglobin, hs-CRP, H-FABP, Lp-PLA2 and PCT. Significantly elevated levels of serum IL-6, IL1β, IL-9 and TNF-α were also recorded in patients with stable angina compared to healthy controls. The results also revealed that patients with stable angina had significantly elevated serum levels of serum triglycerides, total cholesterol, LDL and VLDL with a significant decline of serum HDL compared to healthy controls. We can concluded that, in addition to cTnI, CK-MB and MYO, other biomarkers such as hs-CRP, H-FABP, Lp-PLA2 and PCT are sensitive; and can serve as diagnostic indicators of stable angina pectoris for fast treatment. Furthermore, the detection of inflammatory biomarkers was found to be an additional diagnostic parameter in stable angina.

Quantitative analytics for protein refolding states

Development and optimization of inclusion body (IB) refolding processes is often based on empirical strategies. Thus, generation of in-depth process understanding is crucial to shift towards more knowledge-driven approaches. Within this contribution the goal was to establish a systematic approach for the reliable quantification of process dynamics in protein refolding processes. Established offline- analytical tools like HPLC-based methods or photometric assays were analyzed regarding their specifications and consequent suitability to quantify protein states. An in-silico study was conducted to define requirements for state quantification by analyzing the influences of error, discrete sampling intervals and the pace of the reaction on signal quality. Refolding and aggregation reaction rates were calculated by finite difference approximation with errors propagated from the corresponding folding state. Application of the defined principles on two real Lactate dehydrogenase fed-batch refolding processes resulted in two individual sampling strategies. At reaction rates of up to 0.58 g h−1 description of the ongoing dynamics could be done accurately with high sampling intervals during the first few hours of processing as shown by comparison to a noise free simulation. The proposed method has the potential to be transferred to other proteins and to facilitate the development of model-based monitoring & control strategies.

Green novel photometric and planar chromatographic assays of remdesivir: Comparative greenness assessment study using estimated GAPI tool versus ISO technical reported methods

Abstract Green assessment of analytical procedures has become an environmental obligation in equivalence to their International Council of Harmonization analytical validation obligation. Worldwide awareness of our planet and ecological hazards have raised the shades of green and sustainable chemistry in pure or formulated API assays. The Green Analytical Procedure Index (GAPI) is instant five pentagrams for evaluating the greenness of each step in the developed analytical procedure, in discriminative colors: green, yellow, and red. In the present study, GAPI is applied to assess three novel direct analytical methods: spectrophotometric, fluorimetric, and high-performance thin-layer chromatography (HPTLC) for remdesivir (RDV) quantitation, both in bulk and pharmaceutical vials. Furthermore, a comparative green level calculated GAPI study has been assembled for the proposed methods versus the previously reported methods, for RDV assay, of similar techniques. Spectrophotometric direct Amax method at 240 nm, fluorimetric emission at 404 nm upon excitation at 275 nm as well as the HPTLC densitometric measurement using ethanol and distilled water (7:3, v/v) as mobile phase, all three methodologies are developed, optimized, and fully validated for RDV quantitation. They have been applied to assay RDV pharmaceutical vials and results are compared together with a one-way ANOVA test. Satisfactory recoveries and nano-level sensitivities in addition to the least standard deviations encourage the use of developed methods for routine analysis in quality control laboratories. Their promising greenness profile satisfies the beliefs of ecological sustainability of Green Agenda 2030.

Maize (Zea mays L.) root exudation profiles change in quality and quantity during plant development – A field study

Deciphering root exudate composition of soil-grown plants is considered a crucial step to better understand plant–soil–microbe interactions affecting plant growth performance. In this study, two genotypes of Zea mays L. (WT, rth3) differing in root hair elongation were grown in the field in two substrates (sand, loam) in custom-made, perforated columns inserted into the field plots. Root exudates were collected at different plant developmental stages (BBCH 14, 19, 59, 83) using a soil-hydroponic-hybrid exudation sampling approach. Exudates were characterized by LC-MS based non-targeted metabolomics, as well as by photometric assays targeting total dissolved organic carbon, soluble carbohydrates, proteins, amino acids, and phenolics. Results showed that plant developmental stage was the main driver shaping both the composition and quantity of exuded compounds. Carbon (C) exudation per plant increased with increasing biomass production over time, while C exudation rate per cm² root surface area h−1 decreased with plant maturity. Furthermore, exudation rates were higher in the substrate with lower nutrient mobility (i.e., loam). Surprisingly, we observed higher exudation rates in the root hairless rth3 mutant compared to the root hair-forming WT sibling, though exudate metabolite composition remained similar. Our results highlight the impact of plant developmental stage on the plant–soil–microbe interplay.

Effect of environmental parameters on the content and sugar composition of sulfated polysaccharides in some tropical seagrass

Seagrasses are a paraphyletic group of marine angiosperms that evolved three to four times from land plants and returned to the sea. Halophila ovalis, Thalassia hemprichii and Enhalus acoroides (Hydrocharitaceae) are species that can occur in wide salinity ranges. Sulfated polysaccharides (SPs) comprise a complex group of macromolecules with many critical biological functions. We assume that SP may play a role in salt tolerance in seagrass. In this study, three seagrass species collected in both rainy and dry seasons from the fields were analyzed to determine the total SP contents and different functional groups of SP. Quantification of total SP was done by photometric assays. High-performance anion-exchange chromatography with Pulsed Electrochemical Detection (HPAEC) determined different functional groups of SPs. The results indicated higher total SP contents in seagrass are present in plants at higher salinities and environmental temperatures. The percent of functional groups of SPs are present in the following order: glucose > galactose > arabinose > mannose > rhamnose > fucose. The order is not different between the two seasons.

Fluorimetric microplate assay for the determination of extracellular alkaline phosphatase kinetics and inhibition kinetics in activated sludge

The microbial enzyme alkaline phosphatase contributes to the removal of organic phosphorus compounds from wastewaters. To cope with regulatory threshold values for permitted maximum phosphor concentrations in treated wastewaters, a high activity of this enzyme in the biological treatment stage, e.g., the activated sludge process, is required. To investigate the reaction dynamics of this enzyme, to analyze substrate selectivities, and to identify potential inhibitors, the determination of enzyme kinetics is necessary. A method based on the application of the synthetic fluorogenic substrate 4-methylumbelliferyl phosphate is proven for soils, but not for activated sludges. Here, we adapt this procedure to the latter. The adapted method offers the additional benefit to determine inhibition kinetics. In contrast to conventional photometric assays, no particle removal, e.g., of sludge pellets, is required enabling the analysis of the whole sludge suspension as well as of specific sludge fractions. The high sensitivity of fluorescence detection allows the selection of a wide substrate concentration range for sound modeling of kinetic functions.•Fluorescence array technique for fast and sensitive analysis of high sample numbers•No need for particle separation – analysis of the whole (diluted) sludge suspension•Simultaneous determination of standard and inhibition kinetics

Hydroxy Acid Activation in Fungal Non-Ribosomal Peptide Synthesis Assessed by Site-Directed Mutagenesis.

The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N-methylated l-amino and d-hydroxy acids. They are synthesized by non-ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non-amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.