Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 32; no. 5; pp. 253 - 260
• Main Authors: Bacheva, Anna V., Belyaeva, Anastasiya V., Lysogorskaya, Elena N., Oksenoit, Elena S., Lozinsky, Vladimir I., Filippova, Irina Yu
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.03.2005
• Subjects: Enzymatic peptide synthesis; Low water media; Organic solvents; Subtilisin; Subtilisin immobilized on PVA cryogel; Low water media; Subtilisin; Organic solvents; Subtilisin immobilized on PVA cryogel; Enzymatic peptide synthesis
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2004.12.009

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.