Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

N. P. Balaban^1*, A. M. Mardanova¹, M. R. Sharipova¹, L. A. Gabdrakhmanova¹, E. A. Sokolova¹, G. N. Rudenskaya², and I. B. Leshchinskaya¹

¹Department of Microbiology, Kazan State University, ul. Kremlevskaya 18, Kazan 420008, Russia; E-mail: Nelly.Balaban@ksu.ru

²Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia

^* To whom correspondence should be addressed.

Received May 15, 2002; Revision received April 18, 2003

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A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and K_m and k_cat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

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KEY WORDS: proteinase, thiol-dependent serine proteinase (Bacillus intermedius), purification, properties

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