Streptomyces serine protease (DHP-A) as a new biocatalyst capable of forming chiral intermediates of 1,4-dihydropyridine calcium antagonists.

Streptomyces viridosporus A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4R) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized. Cloning and sequence analysis of the gene for DHP-A (dhpA) revealed that the enzyme was a serine protease that is highly similar in both structural and enzymatic feature to SAM-P45, which is known as a target enzyme of Streptomyces subtilisin inhibitor (SSI), from Streptomyces albogriseolus. In a batch reaction test, DHP-A produced a higher yield of a chiral intermediate of 1,4-dihydropyridine than the commercially available protease P6. Homologous or heterologous expression of dhpA resulted in overproduction of the enzyme in culture supernatants, with 2.4- to 4.2-fold higher specific activities than in the parent S. viridosporus A-914. This indicates that DHP-A is suitable for use in reactions forming chiral intermediates of calcium antagonists and suggests the feasibility of developing DHP-A as a new commercial enzyme for use in the chiral drug industry.

Overexpression and purification of microbial pro-transglutaminase from Streptomyces cinnamoneum and in vitro processing by Streptomyces albogriseolus proteases.

Processing and activation of the precursor of an extracellular Streptomyces transglutaminase were achieved by using three Streptomyces proteases (SAM-P20, SAM-P26 and SAM-P45), all of which are widely distributed in Streptomyces. The use of these proteases would allow us to develop a production process for the active form of this enzyme in recombinant bacteria.