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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. Kostrov2,4

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.