A new nitrilase from Bradyrhizobium japonicum USDA 110. Gene cloning, biochemical characterization and substrate specificity.

A nitrilase gene blr3397 from Bradyrhizobium japonicum USDA110 was cloned and over-expressed in Escherichia coli, and the encoded protein was purified to give a nitrilase with a single band of about 34.5kD on SDS-PAGE. The molecular weight of the holoenzyme was about 340kD as determined by light scattering analysis, suggesting that nitrilase blr3397 self-aggregated to an active form with the native structure being a decamer. The V(max) and K(m) for phenylacetonitrile were 3.15U/mg and 4.36mM, respectively. The catalytic constant k(cat) and specificity constant k(cat)/K(m) were 111min(-1) and 2.6x10(4)min(-1)M(-1). This nitrilase is most active toward the hydrolysis of hydrocinnamonitrile among the tested substrates (4.3 times that of phenylacetonitrile). The nitrilase blr3397 shows higher activity towards the hydrolysis of aliphatic nitriles than that for the aromatic counterparts, and can be characterized as an aliphatic nitrilase in terms of activity. This nitrilase also possesses distinct features from the nitrilase bll6402 of the same microbe.

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