Proteinases immobilized on poly(vinyl alcohol) cryogel: novel biocatalysts for peptide synthesis in organic media

Covalent immobilization of subtilisin and thermolysin on cryogel of poly(vinyl alcohol) was carried out. The biocatalysts obtained are characterized by high stability in water and in DMF-MeCN mixtures of various compositions. The synthetic efficiency of immobilized subtilisin in the multiple iterative synthesis of the peptide Z-Ala-Ala-Leu-Phe-pNA was examined in organic mixtures of different solvent compositions, Immobilized subtilisin exhibits high synthetic activity in organic media. A series of N-acylated p-nitroanilides of tetrapeptides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (Z is benzyloxycarbonyl, Xaa = Leu, Lys, or Glu, Yaa = Phe or Asp; pNA = 4-NO2-C6H4NH-) were synthesized in 70-98% yields using immobilized subtilisin as a biocatalyst without activation and protection of the ionogenic groups of polyfunctional amino acids. Immobilized thermolysin in a DMF-MeCN mixture catalyzed the formation of the peptide Z-Ala-Ala-Leu-pNA, which was obtained in 90% yield (during 1 h). It was demonstrated that the biocatalyst can be used repeatedly and that it retained activity after storage in an aqueous buffer during 6 months.