Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8

Abstract

Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene expressing an inducible nitrilase was located and cloned using degenerate primers coding for well conserved nitrilase sequences, coupled with inverse PCR. The translated sequence showed higher percentage similarity to plant nitrilases than to many bacterial nitrilases. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da and the purified functional enzyme had an apparent molecular weight of ~600 kDa by size exclusion chromatography. In common with the plant nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. Electron microscopy and image classification showed complexes having crescentlike, “c-shaped”, circular and “figure-eight” shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.