Aminoacylase Research Articles

A high-performance liquid chromatography – tandem mass spectrometry – based targeted metabolomics kidney dysfunction marker panel in human urine

BackgroundPrevious studies have examined and documented fluctuations in urine metabolites in response to disease processes and drug toxicity affecting glomerular filtration, tubule cell metabolism, reabsorption, oxidative stress, purine degradation, active secretion and kidney amino acylase activity representative of diminished renal function. However, a high-throughput assay that incorporates metabolites that are surrogate markers for such changes into a kidney dysfunction panel has yet to be described. MethodsA high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of ten metabolites associated with the Krebs cycle, purine degradation, and oxidative stress in human urine was developed and validated. Normal values were assessed in healthy adult (n=120) and pediatric (n=36) individuals. In addition, 9 pediatric renal transplant recipients patients were evaluated before and after initial dosing of the immunosuppressant tacrolimus in a proof-of-concept study. ResultsThe assay met all predefined acceptance criteria. The lower limit of quantification ranged from 0.1 to 1000 μmol/l. Inter-day trueness and imprecisions ranged from 91.4–112.9% and 1.5–12.4%, respectively. The total assay run time was 5.5 minutes.Concentrations of glucose, sorbitol, and trimethylamine oxide (TMAO) were elevated in pediatric renal transplant patients (n=9) prior to transplantation as well as before and immediately after initial dosing of tacrolimus. One month post-transplant urine metabolite patterns matched those of healthy children (n=36). ConclusionsThe LC-MS/MS assay will provide the basis for further large-scale clinical studies to explore these analytes as molecular markers for the patients with renal insufficiency.

Quantification of Hippuric Acid by Stopped-Flow Injection Analysis Using Immobilized Amino Acylase and D-Amino Acid Oxidase in Order to Assay Angiotensin I Converting Enzyme Activity

Quantification of Hippuric Acid by Stopped-Flow Injection Analysis Using Immobilized Amino Acylase and D-Amino Acid Oxidase in Order to Assay Angiotensin I Converting Enzyme Activity

Cloning, Expression & Purification of a Thermostable Aminoacylase from Thermococcus litoralis

Cloning, Expression &amp; Purification of a Thermostable Aminoacylase from <i>Thermococcus litoralis</i>

Hog Kidney and Intestine Aminoacylase-Catalyzed Acylation of L-Methionine in Aqueous Media

Synthesis of N-acyl-L-methionine derivatives was catalyzed by hog kidney and intestinal amino acylase in aqueous media using various acyl donors with acyl-chain lengths ranging from 1 to 6. Various reaction conditions were tested by varying the pH of the buffer, the water-miscible organic solvent and Zn2+ concentrations. Using short chain fatty acids, N-acyl-L-methionine derivatives were efficiently synthesized by both enzymes with a yield of about 20–30%, depending on the acid used. The optimal yield was achieved after a 17-h incubation of the reaction mixture containing 0.02 M acyl sodium salt, 0.2 M L-methionine, 10 μM ZnCl2 and 120 mU of aminoacylase in a 0.2 M sodium phosphate buffer at pH 6.0 and 37°C. The part potentially played by acylase in the in vivo acylation of amino acids, and by extension that of proteins in general, is discussed.

Studies on Immobilization of Aminoacylase From Aspergillus Oryzae on Macroporous Carriers

Studies on Immobilization of Aminoacylase From Aspergillus Oryzae on Macroporous Carriers

Kinetics of Inactivation of Aminoacylase I During Modification of Its Thiol Groups by DPDS and PCMB

The kinetics theory of the substrate reaction during modification of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of aminoacylase I by DPDS and PCMB.From the results obtained we have found that the inactivation reaction of aminoacylase I by DPDS is noncomplexing inhibition,and PCMB reaction is complexing inhibition.The microscopic constants for the reaction of the inactivator with free enzyme and the enzyme-substrate complex were determined.

Immobilization of aminoacylase from Aspergillus oryzae on synthetic modified polyacrylamides.

A series of acrylamide-bisacrylamide copolymers modified by the Mannich Reaction was prepared. The immobilization of aminoacylase from Aspergillus oryzae on the copolymers was studied. All the polymers adsorbed the enzyme and the activity of the immobilized enzyme dependent on the amine used, viz. secondary amine, diamine, or aniline derivative. However, the activity was also influenced by the degree of crosslinking of the polymer. The surface morphology of the dimethylamine-modified polymer, with varying degrees of crosslinking, was analyzed by scanning electron microscope; the polymers having the largest pore diameter possessed the highest enzyme activity. One of the best polymers (DMA-A9B8) was used for immobilization of aminoacylase and its properties were studied. It had high enzymatic activity and good operational stability, i.e., retaining 90% of its original activity after being used for 42 days. The use of these copolymers for the preparation of immobilized enzymes is discussed.

THE IMMOBILIZATION OF AMINOACYLASE AND THE RESOLUTION OF D,L-PHENYLALANINE

A polymer with suitable physical characters and matched functional groups was synthesized,and used as a supporter to immobilize aminoacylase. The results showed that this supporterhad high immobilizing capacity and high selectivity for aminoacylase. Immobilized amino-acylase had high specific activity. In this paper, we determine the physical and chemicalcharacters of aminoacylase for resolution of D,L--phenylalanine, including its optimum tem-perature, pH, ion concentration, activated ion, substrate concentration, thermal stability anddenaturation. The immobilized aminoacylase was used to resolve the D,L-phenylalaninecontinuously. Thc resolved products was separated and purified with ion-exchange resins. L-phenylalanine solution was concentrated by vacuum evaporation and its hydrochloride wasformed. Checking of final product with polarimeter showed high yield and purity.

Anionic exchange nylon laminated membranes for enzyme immobilization

This work presents the optimization of the chemical steps involved in nylon modification with dimethyl sulphate, polyethyleneimine, glutaraldehyde and 2-diethyl aminoethylamine to obtain a weak basic anion exchange support. Activated nylon laminated membranes were utilized for aminoacylase immobilization, allowing an ionically adsorbed enzyme derivative with high activity (0.16 U/mg E·cm2) and low removed activity (<1%). Optimum immobilization conditions and kinetic parameters were also determined. This immobilized enzyme can be used in laminated enzyme membrane reactors.

Thermodynamics of industrially-important, enzyme-catalyzed reactions

The thermodynamics of 10 industrially-important, enzyme-catalyzed reactions are examined. The reactions discussed are: the conversions of penicillin G to 6-amino-penicillinic acid using the enzyme penicillin acylase; starch to glucose using amylases; glucose to fructose using glucose (xylose) isomerase; cellulose to glucose using cellulase; fumaric acid and ammonia to L-aspartic acid using L-aspartase; transcinnamic acid and ammonia to L-phenylalanine using L-phenylalanine ammonia lyase; L-histidine to urocanic acid and ammonia using L-histidine ammonia lyase; lactose to glucose and galactose using lactase; and the reactions catalyzed by amino acylases and proteases. The selection of these processes was based on the economic value of the products and their intrinsic industrial importance. The available thermodynamic properties, such as equilibrium constants, Gibbs energies (delta G degrees), enthalphies (delta H degrees), and heat capacity changes (delta Cp degrees) of these enzyme-catalyzed reactions, are reviewed and summarized. Recommendations are made for future research in this area.

Influence of salts on the covalent immobilization of proteins to modified copolymers of 2-hydroxyethyl methacrylate with ethylene dimethacrylate.

In the study of the covalent immobilization of aminoacylase, thermitase, pepsin, trypsin, chymotrypsin, elastase, subtilisin, penicillinamidohydrolase, carboxypeptidase A, cystathionine-beta-synthase, and anticathepsin D-IgG to copolymers of 2-hydroxyethyl methacrylate and ethylene dimethacrylate (Separon HEMA) containing epoxy groups a marked influence of added salts on the immobilization efficiency was observed. Yields in covalently bound active enzymes were dependent on the concentrations and type of ions added, which can be arranged according to the Hofmeister series. At a distinct concentration, the salting-out ions cause a protein-matrix hydrophobic interaction which is a prerequisite for the covalent bond formation.

Thermostable Aminoacylase fromBacillus thermoglucosidius:Purification and Characterization

Thermostable Aminoacylase from<i>Bacillus thermoglucosidius:</i>Purification and Characterization

Synthesis of the enantiomers of 5-hexadecanolide, the pheromone of the queen of the oriental hornet, vespa orientalis,employing enzymic resolution of (±)-2-aminotridecanoic acid as the key-step

Optical resolution of(±)-2-chloroacetylaminotridecanoic acid with amino acylase gave (S)-2-aminotridecanoic add, which was converted into highly optically pure (S)-5-hexadecanolide (the pheromone of Vespa orientalls). Similarly (R)-2-chloroacetylammotridecanoic acid recovered after the enzymic hydrolysis yielded (R)-5-hexadecanolide. The existing methods for the preparation of the enantiomers of 5-hexadecanolide were critically surveyed to point out basic requisites for an effective chiral synthesis.

The use of immobilized enzymes in the food industry: a review.

The production of high fructose corn syrups was greatly facilitated by the use of immobilized glucose isomerase. Similarly, in Japan, the fermentation industry proved its processing efficiency for amino acids through the use of immobilized amino acid acylase. This article discusses the use of soluble enzymes in the food industry followed by a section on the various available methods to immobilize enzymes. Once enzymes are immobilized, many of their operational parameters could be altered. Rationale for the determination of the effects of immobilization is provided. A relatively new concept is the use of a single matrix for immobilizing more than one enzyme. Immobilized multi-enzyme systems offer many attractive advantages; however, such a process also raises some interesting questions about kinetics. These questions and their suggested answers are discussed in the penultimate section. The major emphasis of this article is on the use of immobilized enzymes in the food industry. Two systems--amino acylase and glucose isomerase--have been demonstrated to be techno-economically feasible. Immobilization of other enzymes, such as glucoamylase, lactase, protease, and flavor modifying enzymes, has received some attention. The potential of these new systems are also discussed.