Delivery of Formulated Industrial Enzymes with Acoustic Technology.

Industrial enzymes are instrumental in many applications, including carbohydrate processing, fabric and household care, biofuels, food, and animal nutrition, among others. Enzymes have to be active and stable not only in harsh application conditions, but also during shipment and storage. In protein stability studies, formulated concentrated enzyme solutions are frequently diluted gravimetrically prior to enzyme activity measurements, making it challenging to move toward more high-throughput techniques using conventional robotic equipment.

Detection of intermediates in the oxidative half-reaction of the FAD-dependent thymidylate synthase from Thermotoga maritima: carbon transfer without covalent pyrimidine activation.

Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity.

Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase.

There are two known types of microbial two-component flavin-dependent monooxygenases that catalyze oxygenation of p-hydroxyphenylacetate (HPA), and they are distinguished by having structurally distinct reductases and oxygenases. This paper presents a detailed analysis of the properties of the enzyme from Pseudomonas aeruginosa, an example of one group, and compares its properties to those published for the Acinetobacter baumannii enzyme, an example of the alternative group. The reductase and oxygenase from P.

Properties of p-hydroxybenzoate hydroxylase when stabilized in its open conformation.

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis.

Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis.

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyses a reaction in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in response to binding p-hydroxybenzoate to the enzyme and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the protein and isoalloxazine ring during catalysis. Earlier research showed that reduction of FAD occurs when the isoalloxazine of the FAD moves to the surface of the protein to allow hydride transfer from NADPH.

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover.

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln.

Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure. In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme.

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide.