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

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 undec1A 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) pLacІ. The recombinant protein was purified to homogeneity. Functional characterization with liquid chromatography-mass spectrometry confirmed that the recombinant Undec1A protein catalyzed the decarboxylation of l-cysteine to form cysteamine.