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Several proteolytic enzymes and dehydrogenases of microbial origin were studied with special regard to structure-activity relationship. Enzyme genes of Zn-proteases, subtilisin and pyroglutamyl aminopeptidase from genus Bacillus, prolyl endopeptidases from Flavobacterium and Aeromonas, and of protease II from E. coli were cloned, sequenced and overproduced in E. coli, their active site structures being elucidated by chemical modification as well as by site-directed mutagenesis. Homology analysis revealed that there is a prolyl endopeptidase family as a new family of serine endopeptidases. In addition, enzymatic properties and the primary structures of glutathione-independent formaldehyde dehydrogenase of Pseudomonas putida and 7 alpha-hydroxysteroid dehydrogenase from E. coli were elucidated. Amino acid sequences deduced from the nucleotide sequences of their genes indicated that the former enzyme should be classified into a long-chain metallo alcohol dehydrogenase family, and the latter belongs to a member of the short-chain nonmetallo-alcohol dehydrogenase family.