Purification and Characterization of a Subtilisin-Like Proteinases Secreted in the Stationary Growth Phase of Bacillus amyloliquefaciens H2

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N. P. Balaban¹, L. A. Malikova¹, A. M. Mardanova^1*, G. N. Rudenskaya², and M. R. Sharipova¹

¹Biological and Soil Science Faculty, Kazan State University, ul. Kremlevskaya 18, 420008 Kazan, Russia; fax: (843) 238-7121; E-mail: Lilianna_kazan@mail.ru

²Chemical Faculty, Lomonosov Moscow State University, 119899 Moscow, Russia; fax: (495) 939-3181; E-mail: laboratoriahps@hotmail.com

^* To whom correspondence should be addressed.

Received November 3, 2006; Revision received December 26, 2006

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Proteinases secreted during the early and late stationary phases have been isolated from the culture liquid of Bacillus amyloliquefaciens H2 using CM-cellulose ion-exchange chromatography with subsequent FPLC on a Mono S column. Considering the character of hydrolysis of specific chromogenic substrates and the type of inhibition, these enzymes were identified as subtilisin-like proteinases. The molecular weight of both proteinases is 29 kD. The proteolytic activity of the proteinases secreted during the early and late stationary phases towards the synthetic substrate Z-Ala-Ala-Leu-pNA was maximal at pH 8.5 and 9.0, respectively. The maximal activity of both proteinases was observed at 37°C, and the proteins were stable within the pH range of 7.2-9.5. The subtilisin-like proteinases from B. amyloliquefaciens were shown to catalyze synthesis of peptide bonds.

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KEY WORDS: subtilisin-like proteinases, Bacillus amyloliquefaciens, purification, properties

DOI: 10.1134/S0006297907040141

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