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

Abstract

BackgroundImmobilization 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.ResultsPorcine 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.ConclusionsBy 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.

Covalent Immobilization of β-Glucosidase into Mesoporous Silica Nanoparticles from Anhydrous Acetone Enhances Its Catalytic Performance

“…One of the first studies on immobilization from a pure organic solvent (n-hexane) concerned lipase [11], whereas other studies investigated the immobilization of enzymes from organic solvents containing small amounts of water (microaqueous organic media). It is claimed that in these kinds of media enzymes become more rigid and then less susceptible to conformational changes [12]. In fact, most enzymes are flexible in water, and covalent immobilization generally leads to their inactivation [13].…”

“…In fact, most enzymes are flexible in water, and covalent immobilization generally leads to their inactivation [13]. Due to their hydrophobic/hydrophilic nature, mostly lipases have been immobilized from organic media [12,14,15], but also other enzymes [12,13]. The performance of the immobilized enzymes resulted in superior activity compared to those immobilized from aqueous media.…”

Papain-Catalyzed Synthesis of Oligolysine in Low-Water Organic Reaction Media

“…In contrast to this, the highly water-soluble basic amino acid oligopeptides do not precipitate and are prone to secondary hydrolysis, leading to poor yields . A few reports have demonstrated the ability of low-water organic media possessing low water activity to support protease-catalyzed synthesis of basic amino acid oligopeptides. , The organic solvents can be introduced in these systems to form either water-miscible homogeneous or water-immiscible heterogeneous systems. The systems utilized a combination of thermodynamically and kinetically controlled synthesis to demonstrate protease-catalyzed oligopeptides synthesis with amino acid esters and amides .…”

Deviation of Trypsin Activity Using Peptide Conformational Imprints

“…However, such enzymes are often immobilized into various substrates to improve stability and reusability without affecting their activity [ 2 , 6 , 7 ]. In this vein, various enzyme immobilization methods have been reported, such as covalent linkage, non-covalent adsorption, and encapsulation systems, among others [ 8 , 9 , 10 , 11 , 12 ]. Nevertheless, the quest for optimum performance is still on due to their conformational changes during immobilization [ 11 , 13 ].…”