Porcine kidney d-amino acid oxidase-derived R-amine oxidases with new substrate specificities.

An R-stereoselective amine oxidase and variants with markedly altered substrate specificity toward (R)-amines were generated from porcine d-amino acid oxidase (pkDAO), based on the X-ray crystallographic analysis of the wild-type enzyme. The new R-amine oxidase, a pkDAO variant (Y228L/R283G), acted on α-MBA and its derivatives, α-ethylbenzylamine, alkylamine, and cyclic secondary amines, totally losing the activities toward the original substrates, d-amino acids. The variant is enantiocomplementary to the flavin-type S-stereoselective amine oxidase variant from Aspergillus niger.

Fabrication of a Polydimethylsiloxane Fluidic Chip Using a Sacrificial Template Made by Fused Deposition Modeling 3D Printing and Application for Flow-injection Analysis.

Fluidic chip fabrication technologies using three-dimensional (3D) printing have received broad attention recently. Herein, we describe a new method for fabricating polydimethylsiloxane (PDMS) fluidic chips using a 3D-printed polyvinyl alcohol (PVA) or acrylonitrile butadiene styrene (ABS) template and polymer coating. In this method, polyethylene glycol (PEG) was coated on the 3D-printed template.

Identification and development of amino acid oxidases.

Amino acid oxidases are an important class of enzymes that mostly participate in the oxidation of amino acids using FAD as a cofactor. Many of them function in the catabolism of amino acids with wider substrate specificities. On the other hand, based on the recent, successful use of the enzymes for diagnoses with new cofactor and mechanism, highly selective enzymes have been screened from Nature, and many new enzymes have been discovered and further characterized by X-ray crystallography.

Expansion of the Substrate Specificity of Porcine Kidney D-Amino Acid Oxidase for -Stereoselective Oxidation of 4-Cl-Benzhydrylamine.

Discovery and development of enzymes for the synthesis of chiral amines have been a hot topic for basic and applied aspects of biocatalysts. Based on our X-ray crystallographic analyses of porcine kidney D-amino acid oxidase (pkDAO) and its variants, we rationally designed a new variant that catalyzed the oxidation of ()-4-Cl-benzhydrylamine (CBHA) from pkDAO and obtained it by functional high-throughput screening with colorimetric assay. The variant I230A/R283G was constructed from the variant R283G which had completely lost the activity for D-amino acids, further gaining new activity toward ()-chiral amines with the bulky substituents.

Origin of Stereoselectivity and Substrate/Ligand Recognition in an FAD-Dependent R-Selective Amine Oxidase.

Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into d-amino acid oxidase from pig kidney.

Low-cost Methods for Making 3D Fluidic Polymer and Glass Chips Using Metal Templates.

Microfluidics is a rapidly growing field in which small volumes of liquid are moved through channels in a large variety of applications. Fabricating such channels can be expensive. Here, we describe an inexpensive method for making 3D channels in fluidic chips by using a sacrificial template made of coated metal wire or metal tubes.

Tailoring D-amino acid oxidase from the pig kidney to R-stereoselective amine oxidase and its use in the deracemization of α-methylbenzylamine.

The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, R-stereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney D-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines.