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

by Chengjian Jiang, Bo Wu
Biochem Biophys Res Commun ()

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.

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