Thiolsubtilisin as an Instrument for Peptide Synthesis. Preparation and Properties

S. V. Kolobanova,¹ E. N. Lysogorskaya,¹ I. Yu. Filippova,¹ V. V. Anisimova,¹ E. S. Oksenoit,¹ and V. M. Stepanov^2,3

¹School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 932-88-46.

²Institute of Genetics and Selection of Industrial Microorganisms, Dorozhnyi proezd 1, Moscow, 113545 Russia; fax: (7-095) 315-05-01; E-mail: ost@vnigen.msk.su

³To whom correspondence should be addressed.

Submitted December 2, 1996.

A convenient procedure for thiolsubtilisin purification from an admixture of subtilisin involving affinity chromatography on bacitracin-Sepharose is presented. Thiolsubtilisin activity was measured by hydrolysis of p-nitrophenyl acetate, a p-nitroanilide-peptide (Glp-Ala-Ala-Leu-pNA), and azocasein. The thiolenzyme catalyzes peptide synthesis. Under these conditions only activated peptide esters, e.g., p-chlorophenyl, N-hydroxysuccinimide, or p-nitrophenyl esters form peptide bonds during interaction with appropriate nucleophiles such as peptides and their derivatives and amino acid amides.

KEY WORDS: subtilisin, thiolsubtilisin, subtiligase, affinity chromatography, bacitracin-Sepharose, enzymatic peptide synthesis.

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