Mechanism and dynamics of fatty acid photodecarboxylase

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

Effect of alkaline protease produced from fish waste as substrate by Bacillus clausii on destaining of blood stained fabric

Alkaline protease or peptidases are the largest groups of enzymes in the biological industry with a variety of application in manufacturing units used in the process of substrate stabilization, dehairing, diagnosis, extraction, food production, destaining, etc., where the pH of the environmental conditions remain above neutral pH. Because of these wider applications of alkaline proteases in industries, their demand is increasing to compete with their chemical counterpart. An alkaline tolerant bacterial strain Bacillus clausii wasisolated from fish waste and used for mass production of alkaline protease using fish waste homogenate as media. Preliminary study on optimization of conditions for the mass production carried out. The optimum temperature for production ranges between 25°C and 35°C and pH determined as 9. The mass production of extracellular alkaline protease carried out using mobilized and immobilized cells of B. clausii at optimized condition using production media, the mixture of production media and fish waste homogenate and in nutrient broth. The recorded results showed that the maximum enzyme production obtained with immobilized cells in nutrient broth media and followed by fish waste homogenate media of 8900 U/ml and 8600 U/ml. Purification protease enzyme yielded 0.35 g/ml from the production media . Bloodstained cloth treated with immobilized enzyme removed the stain completely compared to treatment with non-immobilized enzyme and commercially used detergent. So, the current study suggests the usage of microbial alkaline protease in household detergents to replace the usage of synthetic detergents and save the environment from chemical pollutants.

Site-saturation Mutagenesis of Tyr-105 Reveals Its Importance in Substrate Stabilization and Discrimination in TEM-1 β-Lactamase

The conserved Class A β-lactamase active site residue Tyr-105 was substituted by saturation mutagenesis in TEM-1 β-lactamase fromEscherichia coliin order to clarify its role in enzyme activity and in substrate stabilization and discrimination. Minimum inhibitory concentrations were calculated forE. colicells harboring each Y105Xmutant in the presence of various penicillin and cephalosporin antibiotics. We found that only aromatic residues as well as asparagine replacements conferred highin vivosurvival rates for all substrates tested. At position 105, the small residues alanine and glycine provide weak substrate discrimination as evidenced by the difference in benzylpenicillin hydrolysis relative to cephalothin, two typical penicillin and cephalosporin antibiotics. Kinetic analyses of mutants of interest revealed that the Y105Xreplacements have a greater effect onKmthankcat, highlighting the importance of Tyr-105 in substrate recognition. Finally, by performing a short molecular dynamics study on a restricted set of Y105Xmutants of TEM-1, we found that the strong aromatic bias observed at position 105 in Class A β-lactamases is primarily defined by a structural requirement, selecting planar residues that form a stabilizing wall to the active site. The adopted conformation of residue 105 prevents detrimental steric interactions with the substrate molecule in the active site cavity and provides a rationalization for the strong aromatic bias found in nature at this position among Class A β-lactamases.

Thermodynamics of ß-cyclodextrine/water Interactions

From a purely enthalpic standpoint, the ß-CD hydration, as studied by semiempirical calculations, appears improbable, in contrast to experiment. For the spontaneous hydration process, a compensation mechanism, connected with substrate stabilization and/or entropy increase, is suggested on the basis of simple thermodynamic considerations. The analysis is done on the basis of a single parameter Ew (> 0), which represents the energy needed to transfer a mole of water from the liquid to the ß-CD absorbed phase.The model, although simple, allows predictions largely consistent with the experimental results, while suggesting possible interpretations.