A high-throughput screening method for amino acid dehydrogenase

Anal Biochem. 2016 Feb 15:495:29-31. doi: 10.1016/j.ab.2015.11.012. Epub 2015 Nov 30.

Abstract

A simple and rapid screening method for amino acid dehydrogenase (e.g., leucine dehydrogenase, LDH) has been developed. It relies on a competitive relationship between a non-fluorescent Cu(II)-calcein complex and amino acid (e.g., l-2-aminobutyric acid, l-ABA). When ABA was introduced to a Cu(II)-calcein solution, it bound with the Cu(II) ions and this released calcein from the complex, which was detected as strong fluorescence. The principle of this high-throughput screening method was validated by screening an LDH mutant library. Compared with other methods, this method provided much quicker l-ABA detection and screening for leucine dehydrogenase mutations.

Keywords: Calcein; Cu(II); Leucine dehydrogenase; l-2-aminobutyric acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminobutyrates / metabolism
  • Coordination Complexes / chemistry
  • Copper / chemistry
  • Enzyme Assays / methods*
  • Fluoresceins / chemistry
  • High-Throughput Screening Assays*
  • Ions / chemistry
  • Leucine Dehydrogenase / metabolism*
  • Spectrometry, Fluorescence

Substances

  • Aminobutyrates
  • Coordination Complexes
  • Fluoresceins
  • Ions
  • Copper
  • Leucine Dehydrogenase
  • fluorexon