Molecular cloning and functional characterization of a novel decarboxylase from uncultured microorganisms

Biochem Biophys Res Commun. 2007 Jun 1;357(2):421-6. doi: 10.1016/j.bbrc.2007.03.159. Epub 2007 Apr 3.

Abstract

The metagenomic library approach has been used successfully to isolate novel biocatalyst genes from uncultured microorganisms. We report the cloning of a novel decarboxylase gene by sequence-based screening of a plasmid metagenomic library constructed with DNA from alkaline polluted soils. The gene was named undec1 A and had an open reading frame of 1077 base pairs. It encoded a 359 amino acid polypeptide with a molecular mass of 38 kDa. The predicted protein had 58% similarity to a decarboxylase from Chlorobium phaeobacteroides BS1. The putative decarboxylase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli Tuner (DE3) pLac. The recombinant protein was purified to homogeneity. Functional characterization with liquid chromatography-mass spectrometry confirmed that the recombinant Undec1 A protein catalyzed the decarboxylation of L-cysteine to form cysteamine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carboxy-Lyases / chemistry*
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / metabolism*
  • Cell Culture Techniques
  • Cloning, Molecular / methods*
  • Enzyme Activation
  • Enzyme Stability
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Recombinant Proteins
  • Carboxy-Lyases