Purification and Characterization of Serine Proteinase of the Glu,Asp-Specific Enzyme Family from Thermoactinomyces Species

I. V. Demidyuk,¹ E. A. Nosovskaya,² I. A. Tsaplina,³ G. I. Karavaiko,³ and S. V. Kostrov^2,4

¹Lomonosov Moscow State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 117571 Russia.

²Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 46, Moscow, 123182 Russia; fax: (7-095) 196-21-02.

³Institute of Microbiology, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7/2, Moscow, 117811 Russia.

⁴To whom correspondence should be addressed.

Submitted July 17, 1996.

Enzyme catalyzing hydrolysis of a substrate of Glu,Asp-specific proteinases (Z-Glu-pNA) and cleaving bond Glu13--Ala14 in the oxidized insulin B chain was purified to homogeneity from the culture medium of Thermoactinomyces species using hydrophobic chromatography on phenyl-Sepharose CL 4B as the key purification step. The molecular weight of the proteinase is 23 kD. The enzyme is completely inhibited by diisopropyl fluorophosphate and is stable at pH 5-11. The pH optimum for the hydrolysis of azocasein as substrate is 8.5. The temperature optimum for proteolytic activity is 55°C. The N-terminal sequence of the proteinase is: Ser-Val-Leu-Gly-Thr-Asp-Glu-Arg-Thr-Arg-Val-Thr-Asn-Thr-Thr-Thr-Tyr-Pro-Tyr-Trp-.

KEY WORDS: Glu,Asp-specific serine proteinase, hydrophobic chromatography, purification, characterization.

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