초록
<P>Biocatalytic transformation of carbamate formed readily from CO<SUB>2</SUB> and NH<SUB>3</SUB> provides attractive green routes for mitigation of these important environmental pollutants. Accordingly, a coupled-enzyme system was developed for the one-pot production of citrulline through carbamoylation of ornithine in aqueous solutions of CO<SUB>2</SUB> and NH<SUB>3</SUB>. Hyperthermophilic ornithine carbamoyltransferases are produced recombinantly in <I>E. coli</I> with carbamate kinases known to have a propensity for carbamoyl phosphate synthesis. Importantly, in vitro biocatalysis is carried out by <I>E. coli</I> cell lysate prepared through coexpression of the required recombinant enzymes in a single bacterial culture, greatly reducing limitations normally associated with protein production and purification. Acetate kinase that is endogenous in the lysate also recycles the required ATP cofactor, which would otherwise have been required in costly stoichiometric amounts. Recombinant lysates catalyze the production of carbamoyl phosphate with substoichiometric ATP (>300 turnovers) as well as its in situ reaction with ornithine to give citrulline in high yield (>95%) and g L<SUP>-1</SUP> h<SUP>-1</SUP> titers. The system is active over a wide range of NH<SUB>3</SUB> concentrations (2.5 mM - 2 M), and >90% conversions of NH<SUB>3</SUB> may be reached within 1.5 h. Aqueous NH<SUB>3</SUB> used to sequester CO<SUB>2</SUB> gas (10% v/v) may be directly used as the biocatalyst feedstock. In preliminary studies, citrulline is found to be an effective organic nitrogen fertilizer of the wheat grass <I>Brachypodium distachyon</I>. Therefore, lysates described here constitute a cost-effective biocatalytic platform for one-pot production of a promising organic nitrogen fertilizer, under mild reaction conditions, from environmental pollutants as feedstock.</P><P>An efficient biocatalytic process is developed for the transformation of pollutants, ammonia and carbon dioxide, into a valuable biochemical holding promise as a substitute for urea fertilizer.</P><BR>[FIG OMISSION]</BR>