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Applied and Environmental Microbiology, June 2002, p. 2716-2725, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2716-2725.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Streptomyces Serine Protease (DHP-A) as a New Biocatalyst Capable of Forming Chiral Intermediates of 1,4-Dihydropyridine Calcium Antagonists

Akira Arisawa,1* Motoko Matsufuji,1 Takashi Nakashima,1 Kazuyuki Dobashi,1 Kunio Isshiki,1 Takeo Yoshioka,1 Shigeru Yamada,2 Haruo Momose,2 and Seiichi Taguchi2,3*

Bioresource Laboratories, Mercian Corporation, Fujisawa, Kanagawa 251-0057,1 Department of Biological Science and Technology, Science University of Tokyo, Noda-shi, Chiba 278,,2 School of Agriculture, Meiji University, Kawasaki-shi, Kanagawa 214-8571, Japan3

Received 10 December 2001/ Accepted 20 March 2002

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.

* Corresponding author. Mailing address: Bioresource Laboratories, Mercian Corporation, 4-9-1 Johnan, Fujisawa, Kanagawa 251-0057, Japan. Phone: 81-466-35-1519. Fax: 81-466-35-1524. E-mail: arisawa-a{at}mercian.co.jp.

* Corresponding author. Mailing address: School of Agriculture, Meiji University, 1-1-1 Higashi-santa, Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan. Phone: 81-44-934-7831. Fax: 81-44-934-7831. E-mail: staguchi{at}isc.meiji.ac.jp.

Applied and Environmental Microbiology, June 2002, p. 2716-2725, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2716-2725.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.





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