Improvement of enzymatic performance of Asclepias curassavica L. proteases by immobilization: Application to the synthesis of an antihypertensive peptide

Abstract

The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH. Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Ya) and protein yield (Yp). Asclepain-OGS showed the highest Ya (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Yp was 75 ± 0.4 %. Ya for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.