In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT.

OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1. The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed.     

FREE AMINO ACID POOLS, 15N2 FIXATION AND FIXED N2 ASSIMILATION IN LEUCAENA LEUCOCEPHALA VAR. K-8

Various organs of Leucaena leucocephala (Lam.) de Wit were analyzed for their levels of total nitrogen and free amino acids as well as for changes in free amino acid pools from the time of germination through nodulation. Also an assessment was made of the sink of fixed N₂ (transport product) in the nodules using ¹⁵N methodology. L. leucocephala organs showed total nitrogen levels similar to those of other legumes. Asparagine was the most prevalent amino acid in the nodules and roots followed by glutamate and mimosine. Asparagine was the second most common amino acid in the leaves and stems, with mimosine being the most abundant. Strong correlations were found between the total plant levels of aspartate and glutamate, asparagine and NH₄⁺, acetylene reduction and glutamate, and asparagine and plant age. Asparagine amino- and amide-N accounted for over 75% of the fixed ¹⁵N₂ in nodules. It was concluded that L. leucocephala is an asparagine transporter of fixed N₂ in the nodule.