Activity and Stability of Native and Modified Subtilisins in Various Media

A. V. Bacheva¹, O. V. Baibak², A. V. Belyaeva¹, E. S. Oksenoit¹, T. I. Velichko¹, E. N. Lysogorskaya¹, A. K. Gladilin¹, V. I. Lozinsky², and I. Yu. Filippova^1*

¹Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119992, Russia; fax: (7-095) 932-8846; E-mail: irfilipp@genebee.msu.su

²Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow 119991, Russia; fax: (7-095) 135-5085; E-mail: loz@ineos.ac.ru

^* To whom correspondence should be addressed.

Received December 23, 2002

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The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

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KEY WORDS: immobilized subtilisin, poly(vinyl alcohol) cryogel, complex of subtilisin with poly(acrylic acid), organic solvents

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