Immobilization of trypsin in organic and aqueous media for enzymatic peptide synthesis and hydrolysis reactions

Abstract

Background: Immobilization of enzymes onto different carriers increases enzyme's stability and reusability within biotechnological and pharmaceutical applications. However, some immobilization techniques are associated with loss of enzymatic specificity and/or activity. Possible reasons for this loss are mass transport limitations or structural changes. For this reason an immobilization method must be selected depending on immobilisate's demands. In this work different immobilization media were compared towards the synthetic and hydrolytic activities of immobilized trypsin as model enzyme on magnetic micro-particles.

Results: Porcine trypsin immobilization was carried out in organic and aqueous media with magnetic microparticles. The immobilization conditions in organic solvent were optimized for a peptide synthesis reaction. The highest carrier activity was achieved at 1 % of water (v/v) in dioxane. The resulting immobilizate could be used over ten cycles with activity retention of 90 % in peptide synthesis reaction in 80 % (v/v) ethanol and in hydrolysis reaction with activity retention of 87 % in buffered aqueous solution. Further, the optimized method was applied in peptide synthesis and hydrolysis reactions in comparison to an aqueous immobilization method varying the protein input. The dioxane immobilization method showed a higher activity coupling yield by factor 2 in peptide synthesis with a maximum activity coupling yield of 19.2 % compared to aqueous immobilization. The hydrolysis activity coupling yield displayed a maximum value of 20.4 % in dioxane immobilization method while the aqueous method achieved a maximum value of 38.5 %. Comparing the specific activity yields of the tested immobilization methods revealed maximum values of 5.2 % and 100 % in peptide synthesis and 33.3 % and 87.5 % in hydrolysis reaction for the dioxane and aqueous method, respectively.

Conclusions: By immobilizing trypsin in dioxane, a beneficial effect on the synthetic trypsin activity resilience compared to aqueous immobilization medium was shown. The results indicate a substantial potential of the micro-aqueous organic protease immobilization method for preservation of enzymatic activity during enzyme coupling step. These results may be of substantial interest for enzymatic peptide synthesis reactions at mild conditions with high selectivity in industrial drug production.

Scheme 1

Trypsin-catalyzed conversion of Bz-Arg-OEt to a dipeptide amide and a competing hydrolysis reactions

Fig. 1

Protein coupling yield of trypsin immobilization onto M-PVA in dioxane containing variable water content. Data points were fitted using log normal three parameter equation

Fig. 2

Relative carrier activity of trypsin covalently immobilized onto M-PVA in dioxane containing variable water content. Data points were fitted using log normal three parameter equation

Fig. 3

Reusability of trypsin immobilized onto M-PVA in organic solvent in synthesis reaction (♦) and hydrolysis reaction (▲). One cycle of synthesis reaction corresponds to one hour of peptide synthesis reaction in 80 % ethanol at 20 °C, one cycle of hydrolysis reaction corresponds to 30 min in Tris buffered solution pH 8 at 30 °C. Linear trend lines show possible end values of activity for synthesis (—) and hydrolysis (− − −) reactions

Fig. 4

a Bound protein in g_protein/g_particle and b protein coupling yield as function of protein input for immobilization of trypsin onto M-PVA in dioxane (●) and MES buffer (○)

Fig. 5

Effect of protein input on peptide synthesis activity coupling yield of trypsin immobilized in dioxane (●) and MES buffer (○)

Fig. 6

Effect of protein input on peptide hydrolysis activity coupling yield of trypsin immobilized in dioxane (●) and MES buffer (○)

Fig. 7

Effect of protein input on specific peptide synthesis activity yield of trypsin immobilized in dioxane (●) and MES buffer (○)

Fig. 8

Effect of protein input on specific peptide hydrolysis activity yield of trypsin immobilized in dioxane (●) and MES buffer (○)