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Laccase activity measurement by FTIR spectral fingerprinting
摘要： Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyze the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Laccase activity is commonly determined by monitoring spectrophotometric changes (absorbance) of the product or substrate during the enzymatic reaction. Fourier Transform Infrared Spectroscopy (FTIR) is a fast and versatile technique where spectral evolution profiling, i.e. assessment of the spectral changes of both substrate and products during enzymatic conversion in real time, can be used to assess enzymatic activity when combined with multivariate data analysis. We employed FTIR to monitor enzymatic oxidation of monolignols (sinapyl, coniferyl and p-coumaryl alcohol), sinapic acid, and sinapic aldehyde by four different laccases: three fungal laccases from Trametes versicolor, Trametes villosa and Ganoderma lucidum, respectively, and one bacterial laccase from Meiothermus ruber. By coupling the FTIR measurements with Parallel Factor Analysis (PARAFAC) we established a quantitative assay for assessing laccase activity. By combining PARAFAC modelling with Principal Component Analysis we show the usefulness of this technology as a multivariate tool able to compare and distinguish different laccase reaction patterns. We also demonstrate how the FTIR approach can be used to create a reference system for laccase activity comparison based on a relatively low number of measurements. Such a reference system has potential to function as a high-throughput method for comparing reaction pattern similarities and differences between laccases and hereby identify new and interesting enzyme candidates in large sampling pools.
关键词： FTIR,PARAFAC,enzyme activity assay,spectral evolution profiles,Laccase,high-throughput
更新于2025-09-04 15:30:14
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Characterisation of sensor kinase by CD spectroscopy: golden rules and tips
摘要： This is a review that describes the golden rules and tips on how to characterise the molecular interactions of membrane sensor kinase proteins with ligands using mainly circular dichroism (CD) spectroscopy. CD spectroscopy is essential for this task as any conformational change observed in the far-UV (secondary structures (α-helix, β-strands, poly-proline of type II, β-turns, irregular and folding) and near-UV regions [local environment of the aromatic side-chains of amino acid residues (Phe, Tyr and Trp) and ligands (drugs) and prosthetic groups ( porphyrins, cofactors and coenzymes (FMN, FAD, NAD))] upon ligand addition to the protein can be used to determine qualitatively and quantitatively ligand-binding interactions. Advantages of using CD versus other techniques will be discussed. The difference CD spectra of the protein–ligand mixtures calculated subtracting the spectra of the ligand at various molar ratios can be used to determine the type of conformational changes induced by the ligand in terms of the estimated content of the various elements of protein secondary structure. The highly collimated microbeam and high photon flux of Diamond Light Source B23 beamline for synchrotron radiation circular dichroism (SRCD) enable the use of minimal amount of membrane proteins (7.5 mg for a 0.5 mg/ml solution) for high-throughput screening. Several examples of CD titrations of membrane proteins with a variety of ligands are described herein including the protocol tips that would guide the choice of the appropriate parameters to conduct these titrations by CD/SRCD in the best possible way.
关键词： high-throughput screening,circular dichroism,synchrotron radiation circular dichroism,membrane sensor kinase,ligand-binding interactions
更新于2025-09-04 15:30:14