A novel type of fluorescence resonance energy transfer (FRET) combinatorial libraries were used for the characterization of alkaline serine proteinase produced from Rubrivivax gelatinosus KDDS1. This enzyme was the first serine proteinase characterized from photosynthetic bacteria. The proteinase was found to prefer Met and Phe at the P1 position, Ile and Lys at the P2 position, and Arg and Phe at the P3 position. To date, no serine proteinase has exhibited a preference for Met at the P1 position. Thus, the alkaline serine proteinase from R. gelatinosus KDDS1 is very unique in terms of substrate specificity. A highly sensitive substrate, Boc-Arg-Ile-Met-MCA, was synthesized for kinetic study based on the results reported here. The optimum pH of the enzyme for this substrate was pH 10.7, and the values of kcat, Km, and kcat/Km were 23.7 s(-1), 15.4 microM, and 1.54 microM(-1) s(-1), respectively.