Covalent immobilization of trypsin on glutaraldehyde-activated silica for protein fragmentation

Abstract

Trypsin was immobilized by covalent binding to glutaraldehyde-activated silica with and without a spacer arm; 1,6-diaminohexane and polyethyleneglycol as well. The addition of polyethyleneglycol (PEG) to the immobilization media increased the activity of immobilized trypsin in organic solvents, whilst free trypsin activity disappeared under the same conditions. Thermal, pH, storage, and operational stabilities of the free and immobilized enzyme were found to be better than the free enzyme. Furthermore, use of immobilized enzyme for protein fragmentation was achieved by solid-phase, on-line, protein digestion in organic solvents. Reaction times were reduced to a few minutes and the sample handling was minimized.

Keywords::

• Trypsin
• covalent immobilization
• protein fragmentation in organic solvent

Introduction

Trypsin, which specifically cleaves peptidic bonds on the C-terminal group of lysine or arginine, is a pancreatic serine endoprotease traditionally used for protein digestion and peptide mapping. Digestion with trypsin is generally performed in solution, which presents a number of drawbacks that may limit progress in high-throughput protein identification technology. Long digestion times (typically >5h) are needed because the trypsin-to-substrate ratio has to be kept low to avoid the appearance of interfering autolysis peptides. At low-micromolar substrate concentrations, the production of sufficient peptides to obtain positive protein identification becomes problematic with standard in-solution protocols, since the digestion rate is limited by substrate concentration (Quadroni 1999). The use of long incubation times and elevated temperatures inevitably leads to more digestion artifacts like transpeptidation and non-specific cleavage (Schaefer et al. 2005), deamidation and oxidation (Hunyadi-Gulyas et al. 2004, Lundell and Schreitmuller 1999), and trypsin autolysis products (Karty et al. 2002). Another drawback is manual sample handling and the extra steps that are required for solution-based methods, which can lead to the loss of peptides and the introduction of contaminants like human keratins (Hunyadi-Gulyas et al. 2004).

In view of the strong interest in protein analysis, the concept of immobilized trypsin has received much attention in recent years because of several advantages, such as greater enzyme-to-substrate ratio, high digestion efficiency, possibility of repeated use, and decreased rate of denaturation or inactivation as compared to the free enzyme. In addition, it is easy to remove the enzyme from the substrate solution and prevent contamination of the product (Guibault 1984, Krogh et al. 1999).

The immobilization technique should allow the enzyme to maintain its catalytic activity. Techniques for immobilization have been broadly classified into four categories, namely entrapment, covalent binding, cross-linking, and adsorp tion. It must be emphasized that, in terms of economy, both the activity and the operational stability of the biocatalysts are important during the process. Among these immobilization methods, covalent binding is the most studied because it gives the strongest link, thus providing the most stable polymer–enzyme conjugates without release of the enzyme into solution. However, in order to achieve high levels of bound activity, the amino acid residues essential for catalytic activity must not be involved in covalent linkage to the support. Additionally, it is well known that the characteristics of the support (i.e. shape, particle size, porosity, chemistry, and mechanical strength) may strongly affect basic characteristics of the immobilized enzyme (Bilkova et al. 2005, Podgornik and Tennikova 2002). Therefore, selection of the matrix is a key factor influencing the activity and applicability of the resulting bioreactor. Ideal support properties include physical resistance to compression, hydrophilicity, inertness toward enzymes, ease of derivatization, biocompatibility, resistance to microbial attack, and low cost. Silica-based stationary phases dominate the field of HPLC procedures as a result of the high level of reproducibility, selectivity, and sensitivity that can be achieved. Therefore, it is not surprising that trypsin was first immobilized on silica material more than 20 years ago by Wainer et al. (1989).

Activities of covalently bonded enzymes may be reduced due to conformational changes and decreased accessibility of active sites in the protein structure. Such undesirable conformational effects and steric hindrance can be minimized by the use of spacers to distance the tethered enzymes from the underlying solid surfaces. The introduction of flexible spacers is expected to enhance catalytic activities of the tethered enzymes by offering them greater freedom of movement as well as minimizing unfavorable steric hindrance posed by solid supports (Yuhong and You-Lo 2004).

The use of enzymes in organic solvents greatly expands their potential applications in organic synthesis (Jones 1986). For development of non-aqueous biocatalysts and to make the enzyme soluble in organic solvent, binding of amphiphilic polymers to the enzyme molecules is often applied. Among these polymers, PEG is commonly used to prepare soluble enzyme conjugates. Enzymes modified with polyethylene glycol (PEG), an amphipathic macromolecule, become soluble and active in organic solvents (Yang et al. 1996).

In the field of proteomics, proteins can be identified via peptide fragment identification after proteolysis. Currently, in spite of its limitations, most of these analyses are conducted by means of 2D gel electrophoresis followed by digestion of the proteins, liquid chromatographic (LC) separation, and mass spectrometric (MS) identification of the peptides. However, in the last few years a variety of protocols have been described for the development of trypsin-based bioreactors. Trypsin has been immobilized on different supports based either on silica and polymeric particles and, more recently, on polymeric and silica monolithic materials (Massolini and Calleri 2005). Experiments carried out by using immobilized trypsin have focused on the digestion of model proteins like bovine serum albumin (BSA) (Massolini and Calleri 2005), cytochrome c (Li et al. 2007), and myoglobin (Freije et al. 2005). These studies showed that digestion could be carried out using immobilized trypsin in various format. Common in these experiments is that these substrates were digested in buffered media. However, there was no protein digestion experiment using immobilized trypsin in organic solvent.

In this study, organic solvent-resistant enzyme preparations were prepared by covalent immobilization and immobilization conditions were optimized for characterization of immobilized trypsin. Another goal was to obtain efficient protein digestion and peptide separation in organic solvent. This was realized in a single analytical process using an immobilized enzyme system coupled to HPLC.

Experimental

Trypsin from porcine pancreas (EC 3.4.21.4) (1555 U/mg solid, 88 μg protein/mg solid), N-α-benzoyl-DL-arginine- p-nitroanilide (BAPNA), silicon dioxide (particle size: 0.63-0.2 mm), glutaraldehyde, 1,6-diaminohexane, bovine serum albumin (BSA), and Coomassie brillant blue G250 were purchased from Sigma Chemical Co. (USA). Polyethylene glycol (PEG) 2000 and 5000 were purchased from Fluka (USA). All other chemicals and reagents were of the highest purity. All the experiments were performed in at least triplicate.

Trypsin activity assay

The enzymatic activity of free and immobilized trypsin was measured with a microplate reader (Thermo Scientific) using the chromogenic substrate N-α-benzoyl-DL-arginine-p- nitroanilide, which gives yellow-colored p-nitroaniline (p-NA) upon hydrolysis and can be monitored at 410 nm. The enzymatic activity of free trypsin was measured by hydrolysis of 0.1% BAPNA (10 mg BAPNA, 0.2 mL DMSO, 9.8 mL distilled water) during 10 min at room temperature. The enzymatic activity of immobilized trypsin was measured by hydrolysis of 0.1% BAPNA during 10 min at room temperature and 200 rpm in an orbital shaker. After incubation, immobilized trypsin was removed by centrifugation (4°C, 5 min, 8000 × g). One unit of trypsin activity was expressed as the amount of enzyme that formed 1 µmol of p-nitroaniline per minute under optimum reaction conditions.

Protein determination

Protein concentration of trypsin was determined according to the method of Bradford (1976) with bovine serum albumin as a standard. The amount of bound protein was calculated from the difference between the amount of protein introduced into the coupling reaction mixture and the amount of protein present in the washing water after immobilization. Sample protein concentrations were calculated with calibrations in the range of 20200 µg/mL of bovine serum albumin.

Immobilization of trypsin on silica

Immobilization of trypsin was performed on silicon dioxide (Si-OH) activated with glutaraldehyde by means of a nucleophilic addition reaction. To make immobilized trypsin more flexible on the active silica surface, trypsin was also immobilized by linking with a 1,6-Diaminohexane spacer arm by means of a Schiff base formation. Polyethyleneglycol (PEG 2000 and 5000) was added at a final concentration of 1% to the immobilization media to provide enhanced trypsin activity in organic solvents.

Trypsin immobilization on glutaraldehyde activated silica (Silica-GA-Trypsin)

Silicon dioxide (10 g) was incubated in 100 mL 0.1 M acetate buffer (pH 4.0) containing 1% glutaraldehyde in an orbital shaker at 25°C and 200 rpm for 1 h. The excess glutaraldehyde was removed by washing with distilled water. Activated silica was subsequently incubated in 20 mL trypsin solution (each 1 mL trypsin solution contained 10 mg of enzyme preparation, corresponding to 0.88 mg of protein) and 80 mL 0.1 M phosphate buffer (pH 7.5) at 4°C and 200 rpm for 1 h. Finally, the immobilized enzyme preparation was washed with 0.1 M phosphate buffer (pH 7.5) at least twice.

Trypsin immobilization on activated silica with 1,6-diaminohexane (Silica-GA-DAH-Trypsin)

Glutaraldehyde-actived silica was incubated in 100 mL 0.1 M bicarbonate buffer (pH 8.5) containing 0.1% 1,6-diaminohexane at 25°C, 200 rpm for 2.5 h. The excess 1,6-diaminohexane was removed from the medium by washing with distilled water. DAH-bound support was added to a 100 mL 0.1 M bicarbonate buffer (pH 8.5) containing 1% glutaraldehyde and incubated for 1 h. at 25°C. Excess glutaraldehyde was removed by washing several times with distilled water. DAH-coupled silica support was treated with trypsin as stated.

Trypsin immobilization in the presence of polyethyleneglycol (Silica-GA-PEG-DAH-Trypsin)

Silicon dioxide (10 g) was suspended in 100 mL 0.1 M acetate buffer (pH 4.0) containing 1% glutaraldehyde, 1% PEG 2000 and 5000, and incubated at room temperature in an orbital shaker (200 rpm) for 1 h. Following washing steps, the supports were treated with enzyme. Since PEG masks the protein’s surface, it is expected to show a stabilizing effect against organic solvents for the enzyme. However, if PEG size is increased too much, it might lead to decreased accessibility of the active center. Therefore, PEG 2000 and PEG 5000 were used in this study.

Optimization studies for the immobilization of trypsin

For the three methods used in immobilization of trypsin on silica, different glutaraldehyde concentrations (0.5%, 1%, 1.5%, 2%), and protein amounts (0.8, 1.3, 2.0, 2.6 mg/mL) were tested to determine the optimal conditions for immobilization.

Effect of pH and temperature on the activity of immobilized trypsin

The effect of pH on free and immobilized trypsin activities was estimated with reaction mixtures containing buffers in the range of 4.0 to 11.0. The effect of temperature on trypsin activities was determined at various temperatures (485°C) under optimum conditions.

Effect of organic solvent on free and immobilized trypsin activities

To examine the effect of organic solvent on free and immobilized enzyme activity, an acetonitrile/TFA mix was chosen as a model media for testing the effects in organic solvents, as the separation of peptides produced during protein fragmentation was carried out with acetonitrile gradient elution. Briefly, various portions of acetonitrile / trifluoroacetic acid (30% ACN / 0.1% TFA and 50% ACN / 0.1% TFA) were used to test the enzyme activities in comparison with the buffered medium.

Stability tests and reusability

Thermal stabilities of the both free or immobilized trypsin preparations were determined by measuring the residual activity of the enzyme exposed to 45 and 55°C in 0.1 M sodium phosphate buffer (pH 8.0) for different incubation times (30120 min). In order to determine the pH stabilities, free and immobilized enzymes were incubated at different pH values in 0.1 M buffer solutions at room temperature for 1 h in the range of pH 6.0–12.0 and the remaining activity was measured under standard activity assay conditions.

The storage stabilities of free and immobilized enzyme were estimated by measuring the specific activities after storage at 4°C for a 160-day period, and the remaining activity measurements were performed at 20-day intervals.

Operational stability of the immobilized trypsin was determined for BAPNA in a batch-stirred system. The reaction was carried out for 7 h at 35°C. The activity of samples taken at regular time intervals was measured. The relationship between operation time and the conversion were determined and also the half-life of the biocatalyst was calculated.

Reusability tests were performed at 35°C and were the same as those used for the enzyme activity assay. After each use, immobilized enzyme was removed from the reaction medium and washed with 0.1 M phosphate buffer (pH 8.0).

Protein fragmentation with free and immobilized trypsin

To evaluate the efficacy of protein degradation of free and immobilized trypsin, BSA was chosen as a test protein. Prior to the digestion of BSA, a reduction and alkylation step was performed to denature protein and to increase efficiency of protein degradation. Briefly, 6 M guanidine-HCl and 10 mM 1,4-dithiothreitol (DTT) were added to a 1 mg/mL BSA solution and incubated at 37°C for 1 h, followed by addition of 10 mM iodoacetamide and incubation at 37°C for 15 min. PD-10 desalting column was used after the denaturation process to remove salts. Two systems were established with immobilized trypsin to provide rapid and effective fragmentation of protein and peptide separation. In the first system, protein fragmentation was performed in a solid phase extraction (SPE) module filled with immobilized trypsin following offline separation of protein fragments by HPLC. In the second system, immobilized trypsin was filled in a guard column and connected to the Reverse Phase HPLC system, so that fragmentation and separation were performed simultaneously. The HPLC instrument (Agilent 1100 Series chromatography system) was equipped with a sample loop (100 µL injection volume), a diode-array detector (220 nm wavelength), a thermostat-controlled column oven (50°C), and 250 mm × 4 mm Merck CH-18/2 (5 µm) separation column.

Results and Discussion

Immobilization of trypsin on silica support

Methods of immobilization can affect enzyme activity through the chemical modification of amino acids involved in coupling steps, particularly when the coupling point is situated near the enzyme active site. For this reason, the amount of cross-linker should be optimized. Since glutaraldehyde is a very versatile reagent, covalent immobilization of enzymes by means of glutaraldehyde chemistry is one of the most frequently used technologies for enzyme immobilization (Chang 1971). Among the tested amounts of glutaradehyde, the highest coupling yield and activity of trypsin was observed at 1% glutaraldehyde. For higher molecular weight substrates such as BSA, introduction of an appropriate spacer often confers more flexibility to the enzyme. This can significantly increase the activity of the enzyme, due to reduction of steric hindrance compared with the immobilized enzyme without the spacer.

Different amounts of protein (0.77, 1.25, 2, 2.58 mg) were tested for the investigation of the effect of protein amount on trypsin immobilization performed under the same conditions. Immobilized trypsin activity was decreased regularly with increasing attached protein amount on silica, due to steric hindrance of the existing bound protein so that 0.77 mg protein (10 mg solid/ml) was chosen for the studies. Besides these, 0.1% of 1,6 diamino hexane and 1% of PEG 2000 or PEG 5000 was chosen for enzyme immobilization. Optimized immobilization yields are summarized in Table I.

Table I. Immobilization of trypsin on silica support

		Specific activity^a (nmol/min/mg)
Immobilization method	Immobilization yield (%)	A	B	C	Optimum temperature ˚C	Optimum pH
Silica-GA	63	92	106	90	45	9.5
Silica-GA-Dah	60	103	115	97	45	9.5
Silica-GA- PEG2000	50	114	118	111	45	10
Silica-GA- PEG5000	63	79	102	95	45	10

^a Specific activity of immobilized trypsin in A: 0.1 M phosphate buffer (pH 8); B: 30% ACN + 0.1% TFA; C: 50% ACN + 0.1% TFA.

Effect of temperature and pH on free and immobilized trypsin activity

The maximum activity was observed at 45°C for both forms of enzyme and, generally, the optimum temperatures of animal-origin trypsin can typically be found in the range of 45°C (Guyonnet et al. 1999, Ahsan and Watabe 2001, Kishimura and Hayashi 2002).

pH is one of the most important factors influencing not only the properties of amino acid side groups but also the solution chemistry of the insoluble support. Thus, protein–support interaction and surface properties of a protein are strongly influenced by the pH. While the optimum pH value of free trypsin was determined at pH 8.0, the immobilized trypsin was most active in the range of pH 9.0 to 10.0. These results were expected because the number of positively charged groups of enzyme linked with the amino groups to the carrier decreases after immobilization. Thus the character of the enzyme becomes more polyanionic (Vasudevan et al. 2004). In general, the optimum pH values of animal-origin trypsin are in the range of pH 7.0 to 9.0 (Kishimura and Hayashi 2002, Fengna et al. 2004).

Effect of organic solvent on free and immobilized trypsin activity

The use of enzymes in organic solvents greatly expands their potential applications in many areas like online protein fragmentation for HPLC separation. However, many enzymes suffer substantial losses in activity in organic solvents. This study was designed to prepare immobilized trypsin for protein fragmentation and simultaneous online HPLC separation by using organic solvent gradient. Stability in organic solvents is therefore a highly desirable characteristic for this application. Data showed that the free enzyme has no activity in the organic solvent, whereas the immobilized enzyme had better activity in organic solvents in comparison with the buffered medium (Table I).

Stability of free and immobilized trypsin

Immobilization often stabilizes the tertiary structure of enzymes, thereby allowing their application under even harsh environmental conditions of pH, temperature, and organic solvents, and thus enables their use at optimum performance. Stability of enzyme preparations are the time-dependent protection of enzyme activity in certain operating conditions. As a result of immobilization, there is a general increase in the stability of the enzyme.

One of the most important parameters which affects the stability of enzymes is temperature. Enzymes are usually more stable at lower temperatures while rapid thermal denaturation occurs at high temperatures. Time-dependent thermal stability of free and immobilized trypsin was determined at 45 and 55°C (Figure 1).

Figure 1. Thermal stability of free (♦) and immobilized trypsin [(▪) Silica-GA-Trypsin ; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin] at 45°C (A) and 55°C (B).

The immobilized enzyme was found to be more thermostable than the free enzyme. Immobilized enzyme and free enzyme retained 65% and 45% of activity, respectively, at 45°C after 2 h. At 55°C, immobilized enzymes retained 40% of activity, whilst the free enzyme retained 18% activity after 2h. These results showed that the thermostability of the immobilized trypsin improved considerably as a result of immobilization. In several studies of bovine pancreatic trypsin, the enzyme preserves a large proportion of activity until 50°C, after which it rapidly loses activity above this temperature (Fernandez et al. 2005, Montero et al. 2007).

The immobilized trypsin was found to be stable across a pH range of 7.0–11.0, while the free enzyme showed more limited stability in the pH range of 7.0–9.0 (Figure 2). It was determined that the immobilization process increased enzyme pH stability. In general, free trypsin of animal origin is stable in the range of pH 6.0–9.0 (Kishimura and Hayashi 2002, Fengna et al. 2004), while the same enzyme immobilized on chitosan-bead-supported silica gel showed higher stability in the basic region (Fengna et al. 2004).

Figure 2. pH stability of free (♦) and immobilized trypsin [(▪) Silica-GA-Trypsin; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin].

The high cost of enzymes used for industrial purposes and the time necessary for their immobilization have led to increasing interest in the storage stability of these enzymes for longer periods. Storage stability is an important advantage of immobilized enzymes over the free enzymes, because free enzymes can quickly lose their activity. Free and immobilized trypsin were stored at 4°C under the same conditions and the activity measurements were carried out for a 160-day period. Under the same storage conditions, the free enzyme was found to lose about 40% of its initial activity over this 160-day period, whereas the immobilized enzyme lost only 10% of its initial activity over the same period of time (Figure 3).

Figure 3. Storage stability of free (♦) and immobilized trypsin [(▪) Silica-GA-Trypsin; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin].

The decrease in activity was explained as a time-dependent natural loss in enzyme activity, and this was prevented to a significant degree by immobilization. The experiment revealed that storage stability of the immobilized trypsin was improved compared to free enzyme, and 4°C is a suitable temperature for storage.

Operational stability of immobilized trypsin is shown in Figure 4.

Figure 4. Operational stability of immobilized trypsin [(▪) Silica-GA-Trypsin; (▲) Silica-GA-Dah-Trypsin; (•) Silica-GA-PEG2000-Trypsin; (*) Silica-GA-PEG5000-Trypsin].

Operational stability is a parameter that is demonstrated in the change of enzyme activity during the study period. Catalytic potential of an enzyme is proportional to operational stability. Often, enzyme stability is expressed in terms of half-life, which is the time after which enzyme activity decreased to 50% of initial activity. Determination of half-life was calculated by the following equations:

t_1/2 =0,693k_D k_D =2,303/ t x log(A₀/A)

where t equals duration of operation, and k_D the decay coefficient. Activity (A) and intial activity (A₀) were measured, respectively.

The half-lives (t_1/2) of immobilized Silica-GA-Trypsin, Silica-GA-Dah-Trypsin, Silica-GA- Peg2000-Trypsin, Silica-GA-Peg5000-Trypsin were found to be 141, 77, 126, and 350 minutes, respectively.

Among the immobilization methods, Silica-GA-Dah-Peg5000-Trypsin had the best operational stability with respect to half-life. The PEG polymer has also been reported to have a stabilizing effect on certain enzymes to which it has been linked (Manta et al. 2003), PEGylation has previously been found to increase the thermodynamic stability of protease (Rodríguez-Martínez et al. 2008). PEG conjugation masks the protein’s surface and increases the molecular size of the polypeptide, thus reducing the degradation by proteolytic enzymes.

Re-usability of immobilized trypsin

One of the most important goals of enzyme immobilization is recycling and reuse of the enzyme after use in the process. The re-usability of the immobilized enzyme in organic solvents was investigated for this purpose. Thus re-usability trials were carried out in the appropriate buffer solution (0.1 M phosphate buffer, pH 8.0), as well as with different ratios of organic solvents (30% ACN +0.1% TFA and 50% ACN +0.1% TFA). Activity of the same enzyme preparations was measured eight times per day, and the activity measurements were continued for three days. Different immobilization methods were compared with each other, and as a result of repeated use, enzymes were found to give almost parallel results with each other. As a result of immobilizion, enzyme activity in the buffer was found to yield 20% of remaining enzyme activity after 24 repetitions. However, upon re-use in different organic solvents, a rapid loss of enzyme activity was determined after the initial use. Although the first activity measurement in organic solvent was comparable to that in buffered media showed high activity (Table I). It is known that the digestion efficiency of proteins using immobilized trypsin can be enhanced by the use of organic solvents during digestion. This is because proteins tend to denature in the presence of organic solvents like acetonitrile, thereby increasing cleavage-site accessibility (Slysz and Schriemer 2003). However, organic solvent studies carried out with the free enzyme showed no activity in any percentage of organic solvents. These results emphasize that maintaining activity of immobilized enzymes in organic solvents is of particular importance.

Protein fragmentation with free and immobilized trypsin

In comparison to chemical digestion, enzymatic digestion is popular due to its versatility. The routine proteolysis of proteins is performed in solution, but it suffers from drawbacks, such as long incubation time, enzyme auto-digestion, and manual manipulation. Trypsin is one of the least stable neutral proteases. Its rapid autolysis in solution makes it difficult to control the reaction conditions and, as a consequence, the catalytic efficiency of this enzyme is decreased and its cost has increased. Figure 5 shows the peptide chromatogram of products obtained from free trypsin (A), solid phase extraction (B), and on-line digestion (C), respectively.

Figure 5. BSA fragmentation with: A: free trypsin (overnight degradation in buffered solution); B: immobilized trypsin-solid phase module (15 min degradation in 30% ACN); and C: online trypsin reactor coupled with HPLC (30 min online degradation in column with 0.01 mL/min flow rate) [HPLC conditions for peptide separation: Flow rate: 0.4mL/min; Column temperature: 50°C; Mobile phases A: 0.1% TFA, B: ACN; Elution: 1%-50% ACN gradient; Wavelength: 220nm].

According to the results of stability tests, the immobilization method of Silica-GA-Peg5000-Trypsin was selected to examine efficiency of the immobilized enzyme in protein degradation. Prior to the degradation of bovine serum albumin, reduction and alkylation steps were performed to increase efficiency of protein degradation.

As is seen, all analyses have complex chromatograms with many peaks; as a result, immobilized trypsin digestion shows more and higher intensity peaks than with free trypsin. Digestion of BSA using the on-line immobilized trypsin reactor is more effective than the digestion of BSA using a solid phase extraction reactor in terms of peak intensity. A good correlation between these two systems was observed in relation to digestion times. An interesting finding common in articles dealing with trypsin digestion through immobilized systems is the increase in the amount of low-molecular-weight peptide fraction (Calleri et al. 2004, Ota et al. 2007), which is a consequence of the immobilized trypsin catalytic mechanism. This was also observed in both chromatograms (Figure 5B and C). When comparing protein degradation performed with immobilized enzyme and free enzyme, we observed that immobilization of trypsin is essential for effective and time-efficient peptide fragmentation. To speed up and automate protein degradation and peptide separation, this online method was established by immobilizing trypsin on solid supports to perform solid-phase protein digestion and peptide separation in organic solvent. As a result of this, digestion was performed in flow-through conditions with reduction in reaction times and with an increased peptide yield. It should be emphasized that the use of immobilized trypsin decreased the digestion time from overnight to around 30 min. As a result of this immobilization, sample handling is minimized because of the direct connection of the enzymatic reactor to the analytical system. These results indicate that this system can be used in conjunction with mass spectrometry, for peptide mapping based on the separation and identification by MS of peptides generated by proteolytic digestion.

Conclusions

In this study, immobilization of trypsin was successfully carried out on a silica-based carrier using different methods. The immobilized enzyme with good stabilities and reusability increases the potential use of trypsin for different biotechnological applications. According to the results obtained after optimization and characterization of the immobilized enzyme, the potential use of immobilized enzyme preparations for protein fragmentation was investigated. These results emphasize that the immobilization process offers considerable advantages for process time and peptide efficiency. Significantly, high digestion efficiency was obtained with short contact times. Furthermore, the tryptic reactor showed that protein separation and digestion can be integrated in organic solvent. This study also demonstrates that immobilization of enzymes are becoming much more important, especially within organic solvents. The combined properties of the immobilized enzyme make it a good candidate for effective protein degradation and further development of this method can enable an extension of its application towards peptide mapping.

Acknowledgements

The authors would like to thank Professor U.T. Bornscheuer (University of Greifswald) for providing the opportunity to work in his laboratory during part of this study. This work was partially supported by the Scientific Research Foundation of Ege University, Izmir, Turkey (BAP-Grant Number Sci-2010-003).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.