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 P-1 position, Ile and Lys at the P-2 position, and Arg and Phe at the P-3 position. To date, no serine proteinase has exhibited a preference for Met at the P-1 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 K-cat, K-m, and k(cat)/K-m were 23.7 s(-1), 15.5 muM, and 1.54 muM(-1) s(-1), respectively. (C) 2003 Elsevier Inc. All rights reserved.