Immobilization of trypsin in organic and aqueous media for enzymatic peptide synthesis and hydrolysis reactions
© Stolarow et al. 2015
Received: 20 February 2015
Accepted: 12 August 2015
Published: 19 August 2015
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.
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.
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.
Enzyme immobilization is a method suitable for enzyme reutilization and stabilization possibly leading to lower process costs . However, the number of different immobilization techniques, as well as enzyme carriers, is diverse and complicating. The choice for the best suitable carrier as well as immobilization method has to be done depending on the immobilizate’s application.
Non-porous magnetic microparticles feature a good superparamagnetic separation characteristics, as well as a relatively small particle size of approximately 1 μm, which makes them less prone to mass transport limitations . Due to the particle size, the specific surface available for enzyme coupling is increased compared to other non-porous supports .
Micro-aqueous organic media have beneficial effects on enzymatic stability in synthesis reactions where the enzymes may become more rigid and thus less susceptible to conformational changes in the media . Proteins have been reported to show a “pH-memory” of the last aqueous medium prior to lyophilization and are able to maintain their ionization state in micro-aqueous organic solvents [5, 6]. When lyophilized from a pH corresponding to the optimum pH, the activity of the lyophilized and organic solvent resubstituted enzyme has been described to be at its maximum . These effects have been investigated among different enzymes catalyzing synthesis reactions in organic media [7–9].
Trypsin catalyzes hydrolysis reactions, as well as peptide synthesis reactions, when the equilibrium is pushed on either hydrolysis or synthesis product side [10, 11]. This process of protease usage in peptide synthesis and modification reactions is often assumed to offer a more selective and environmentally-friendly option for peptide-hormones and other oligo-peptide-based bio-active agents compared to chemical solid-phase syntheses . Therefore the usage of immobilized trypsin for these kinds of reactions is desirable in order to lower the costs of the process. The application of organic solvent systems for immobilization of hydrolases is so far limited to few publications. One of the first investigations on immobilization in organic solvent was conducted for lipase . The researchers compared aqueous, microemulsion and organic systems for immobilization to test the lipase activity in a hydrolysis and transesterification reactions. Within transesterification reaction the organic and microemulsion systems showed 20 and 40 % residual activities, respectively, for immobilization while within aqueous method no transesterification activity after immobilization could be detected. In subsequent works lipase from Candida rugosa was immobilized in apolar and polar solvents such as hexane and acetone and compared to aqueous immobilization in buffer [14, 15]. The results showed a sevenfold higher specific activity of acetone immobilized lipase in butyl butyrate synthesis reaction compared to aqueous solution immobilization. Sun et al.  immobilized lipase from Candida antarctica by adsorption in isooctane. This immobilization achieved an up to threefold higher activity recovery compared to adsorption in aqueous medium. Zhu et al.  immobilized several enzymes including trypsin within micro-aqueous organic media onto chitosan microspheres. Immobilization under micro-aqueous conditions showed a twofold higher remaining catalytic activity compared to water phase immobilization, which appears to be a promising alternative to the mostly used aqueous immobilizations.
In the present study an immobilization method in organic solvent was analyzed considering different immobilization parameters influencing the protein coupling and activity coupling. The enzymatic activity was measured in a dipeptide synthesis reaction. The operational stability was determined in ten sequential peptide synthesis and hydrolysis cycles. Furthermore, the elaborated immobilization method was compared to an aqueous media immobilized trypsin by such factors as protein coupling yield, activity coupling yield, specific activity yield. A special focus was set on the comparison of these immobilization methods in both possible trypsin-catalyzed reaction types: the hydrolysis reaction which is performed at physiological conditions in aqueous medium and synthesis reaction in organic medium which plays an important role in industrial drug production.
Results and discussion
Enzymatic synthesis of Bz-Arg-Arg-NH2
Screening for suitable solvent and parameter optimization for enzyme immobilization
Relative carrier activity for trypsin immobilization in different organic solvents with various physical properties
Relative carrier activity/%
Log KOW a
Influence of water content in dioxane on protein coupling and carrier activity during immobilization
Reusability of trypsin immobilized in dioxane
Comparison of dioxane and aqueous immobilization methods for peptide hydrolysis and synthesis
In the present study a method for covalent trypsin immobilization on magnetic particles in organic solvent was investigated. The used magnetic particles were stable within several organic media and suitable for trypsin immobilization. Trypsin immobilized within dioxane solvent conditions showed a higher specific activity and activity coupling yield in peptide synthesis reaction. For peptide hydrolysis the aqueous method achieved better results regarding activity coupling yield over a protein input range of 0.001–0.05 gprotein/gparticle, but was similar to dioxane immobilization in specific activity yield. These results indicate a substantial potential of the organic protease immobilization method for preservation of enzymatic activity during enzyme coupling step. The results revealed, that the organic method suffers from enzyme solubility limitations at given conditions. Consequently, future investigations should focus on improvement of protein solubility for immobilization in organic medium and testing of protease immobilization in water-immiscible organic solvents.
Magnetic poly(vinyl alcohol) micro-particles (M-PVA) C22–C250 were provided by PerkinElmer Chemagen, Baesweiler, Germany. The used beads have a carboxyl functionalization with a concentration of 950 μmol COOH/gparticle and particle size distribution within 1–3 μm. 1,4-dioxane, 1-propanol, methanol, ethanol, acetone, acetonitrile, 2-(N-morpholino)ethanesulfonic acid (MES), carbonyldiimidazol (CDI), calcium chloride, tris(hydroxymethyl)aminomethane (Tris) and trifluoroacetic acid (TFA) were purchased from Carl Roth, Karlsruhe, Germany. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), Nα-Benzoyl-L-arginine ethyl ester hydrochloride (Bz-Arg-OEt), Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA), p-nitroaniline (pNA) and L-arginine amide dihydrochloride (Arg-NH2) were purchased from Sigma-Aldrich, Deisenhofen, Germany. Recombinant porcine trypsin (EC 18.104.22.168) was purchased from Roche Diagnostics, Mannheim, Germany. Nα-Benzoyl-arginyl-arginine amide trifluoroacetate (Bz-Arg-Arg-NH2) was synthesized by Bachem, Weil am Rhein, Germany. Nα-Benzoyl-L-arginine (Bz-Arg) was obtained from ABCR GmbH, Karlsruhe, Germany. BC Assay was performed by a BC Assay Protein Quantitation Kit from Interchim, Uptima, Montluçon, France. HPLC analysis was performed with a standard HPLC device from Agilent 1200 Series, Agilent, Waldbronn, Germany equipped with a reversed phase column (C18 Luna, 5 μm, 250 × 4.60 mm, Phenomenex, California, USA).
Covalent immobilization of trypsin in organic and aqueous media
For immobilization in organic media the protocol was as follows. The M-PVA particle suspension concentration was adjusted to 25 mg/ml and washed with 0.1 M MES buffer pH 5.3 for three times followed by four washes in dried 1,4-dioxane with >99.8 % purity. For activation of functional COOH-groups on particle surface CDI was used as activating substance at 0.12 gCDI/gparticle and incubated with particles at 18 °C for 82 min. After activation particles were washed with 1.4-dioxane for two times. Trypsin was lyophilized at pH 7 prior to immobilization, suspended in dioxane with given water content from 0 to 10 % at concentrations of 0.002–0.080 mg/gparticle and incubated at room temperature for 1 h.
For immobilization in aqueous media a modified protocol according to Hermanson (2008)  was applied. The pH 5.3 adjusted 25 mg/ml particle suspension was activated by a 1.6 gEDC/gparticle EDC solution for 6 min at 11 °C and washed two times with 0.1 M MES buffer pH 7. Trypsin solutions in a range of 0.002–0.080 g/gparticle in 0.1 M MES pH 7 were added to the particles and incubated for 30 min at 25 °C for enzyme coupling.
After enzyme coupling step the particles from each immobilization method were washed with 0.04 M Tris buffer pH 9 for three times and stored in 0.02 M CaCl2 solution at 4 °C. The coupling supernatant and washing solutions were used for quantification of the bound enzyme by BC Assay. The particle concentration after immobilization was determined gravimetrically by drying 250 μl of the respective immobilizate at 70 °C for at least 16 h in micro-tubes.
Synthetic and hydrolytic activity measurement of free and immobilized trypsin
For measurement of synthetic trypsin activity 200 μl of a 25 mg/ml immobilizate suspension were used removing the supernatant by magnetization. 1.5 ml of a substrate solution containing 10 mM Bz-Arg-OEt, 200 mM Arg-NH2 in 80 % (v/v) ethanol and 20 % 0.1 M Tris buffer pH 9 with 0.02 M CaCl2 was added to the immobilizates starting the synthesis reaction. Samples were taken without particle uptake and mixed in a 1 to 4-ratio with 6 % (v/v) TFA in order to terminate the reaction. For free trypsin an enzyme concentration of 0.1 mg/ml in 1.5 ml substrate solution was used. Samples were analyzed by HPLC. 1 Unit is defined as 1 μmol of synthesized Bz-Arg-Arg-NH2/min of reaction.
Hydrolytic activity determination was accomplished using a 1 mM BAPNA solution in 0.1 M Tris buffer pH 8 containing 0.02 M CaCl2. The reaction was conducted at 30 °C and initiated by introduction of 1.5 ml of substrate solution to 200 μl particles without supernatant. Samples were taken and the reaction was terminated by a 1 to 4-ratio of sample to 6 % TFA solution. For determination of free enzyme activity a solution of 0.01 mgtrypsin/ml in 1.5 ml of substrate was used. Samples as well as pNA standard were analyzed in a microplate reader at 405 nm. 1 Unit is defined as 1 μmol of pNA released per minute of hydrolysis reaction.
Protein quantification assay
Protein quantification of protein stock solutions and supernatants was performed using a standard BC Assay Protein Quantitation Kit. The assay was used in order to quantify the protein loading of immobilized enzyme measured against bovine serum albumin standard in the respective buffer solution.
High pressure liquid chromatography (HPLC) analysis of peptide synthesis samples
Peptide synthesis samples were analyzed by RP-HPLC-UV/Vis in a 18 %/82 % (v/v) ACN/H2O solution as mobile phase containing 0.05 % TFA, at a column temperature of 60 °C for 13 min at 254 nm. As standard substances Bz-Arg-OEt, Bz-Arg, and Bz-Arg-Arg-NH2 were used in order to quantify the product concentrations.
This work was partially funded by the German Federal Ministry of Education and Research (FKZ: 0315815). Sanofi-Aventis Deutschland GmbH is acknowledged for financial support.
All figures have been created by SigmaPlot software version 12.5 (Systat Software Inc., San Jose, CA, USA) and are attached to this manuscript.
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