Detecting and assaying protein-protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein-protein interaction, i.e. firefly luminescent intermediate-based protein-protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a "Donor" that catalyzes the adenylation of firefly luciferin to produce a luciferyl-adenylate intermediate, and an "Acceptor" that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl-adenylate formed during the catalysis and an increased ATP-based Km value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.

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Two enzymes, nitrile hydratase and amidase, which participate in the conversion of trans-1,4dicyanocyclohexane (f-DCC) to fra/is-4-cyanocyclohexane-l-carboxylic acid (f-MCC), a tranexamic acid intermediate, were purified and characterized. Nitrile hydratase was obtained in a homogeneous state. The molecular weight of the native enzyme was 61,400 and that of the subunit 26.900, indicating a dimer structure. Valeronitrile and butyronitrile were good substrates for the enzyme. The enzyme could also hydrate benzonitrile, /j-hydroxybenzonitrile and 4-cyanobenzoic acid. f-DCC was exclusively hydrated to fra/is-4-cyanocyclohexane-l-carboxyamide (f-MCMA), further hydration of the nitrile group of r-MCMAand t-MCCnot being observed. The presence of pyrroloquinoline quinone in the enzymewas confirmed. The presence of iron was also confirmed. The amidase of the strain was also purified. The latter enzyme could hydrate f-MCMA,yielding f-MCC. The enzyme was highly resistant

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