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. 2025 Aug 22;10(35):40534–40543. doi: 10.1021/acsomega.5c06073

Liposomes Carrying Surface-Conjugated Trypsin for Controlled Proteolysis Reactions

Mikiya Wakabayashi 1, Noriko Yoshimoto 1, Makoto Yoshimoto 1,*
PMCID: PMC12423795  PMID: 40949228

Abstract

Control of the proteolytic activity of trypsin is an important challenge in proteomics and food engineering. In this work, trypsin was covalently conjugated to lipid membranes possessing poly­(ethylene glycol) in a 0.1 M sodium phosphate buffer solution (pH = 7.0) to control the enzyme functions using a liposomal environment. The amount of trypsin conjugated per liposome membrane area was maximized based on the size of the polymer chain of liposomes and the type of cross-linker (glutaraldehyde or tris-succinimidyl aminotriacetate). The secondary structure of trypsin was altered through the conjugation reaction, as revealed with circular dichroism measurements and spectral analyses. Stability of trypsin at 40–60 °C significantly increased through being conjugated to liposomes, whereas free trypsin was clearly deactivated even at 40 °C in an enzyme concentration-dependent manner, indicating the involvement of autolysis mechanism. Liposome-conjugated trypsin was applied to catalyze the digestion of bovine serum albumin at 37 °C and a substrate/trypsin weight ratio of 100. The proteolysis rate observed with liposome-conjugated trypsin was smaller than that of the free enzyme, while the rate clearly depended on the characteristics of liposomes. The results obtained demonstrate that liposomes carrying surface-conjugated trypsin are biocompatible, heat stable, and reactivity-controllable catalysts for proteolysis reactions.

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1. Introduction

Trypsin is a serine protease and one of the essential enzymes for proteomics analyses, − weakening the adhesive interaction between biomolecules and solid surface, , and quality control of dairy products like cheese and milk. − Diverse methods for preparing water-insoluble carrier material-bound trypsin − or functional molecule-modified trypsin , have been reported, providing opportunities to perform above tryptic reaction-based operations in a stable and controlled manner. One of the major applications of immobilized trypsin is the digestion of proteins in batch or flow-through reactors. − ,,, These approaches potentially allowed for a high local trypsin/proteinaceous substrate ratio to be maintained while minimizing enzyme autolysis and contamination of a reaction mixture. On the other hand, when trypsin is applied to food processing , or adherent cell culture, it is necessary to moderately control the reactivity of trypsin in order to maintain necessary function and structure of cells or biomolecules after tryptic treatments. , Furthermore, sufficient biocompatibility of carrier materials employed for trypsin has become of primary importance for biotechnological and food engineering applications.

Phospholipid vesicles (liposomes) carrying proteinases have been prepared through confinement in internal aqueous phase of liposomes. − Trypsin-containing liposomes were prepared for detecting membrane permeation of a peptide based on its enzymatic cleavage inside liposomes. A different type of liposomes containing encapsulated trypsin was also applied to cheese-ripening process. , On the other hand, liposomes modified with a ligand were used to capture and purify trypsin based on ligand affinity chromatography. This approach gave liposomes carrying surface-bound trypsin. Furthermore, hydrophobized trypsin could be incorporated in lipid bilayer membranes of liposomes, although, to the best of our knowledge, little is known so far concerning the optimal preparation, autolysis, and proteolysis activity of liposome-bound trypsin.

In our previous work, a cysteine protease, papain was covalently conjugated to poly­(ethylene glycol)-tethered liposomes via a cross-linker, and the conformational structure and stability of the conjugated enzyme were examined. The secondary structure of papain was maintained at high temperatures in a lipidic environment. As a result, the heat stability of liposome-conjugated papain tended to increase compared to that of free papain, as examined in the absence of an enzyme activation reagent (cysteine) at heating. Liposome-papain conjugates were applicable to catalytic digestion of antibodies, and the liposomal catalyst was advantageous to be separated from a digest with size-exclusion chromatography.

In the present work, trypsin was conjugated to preformed liposomes as highly biocompatible and self-dispersible enzyme carriers to realize flexible control of the autolysis and proteolysis reactions based on the size of the poly­(ethylene glycol) chain and enzyme density on the surface of liposomes. Heat-induced acceleration of autolysis was examined in detail with respect to free trypsin to evaluate the role of liposomes on the stability of surface-conjugated trypsin. The proteolytic activity of various types of liposomes carrying conjugated trypsin was also examined with bovine serum albumin as a model substrate to explore the liposomal-conjugation-driven functionality of trypsin.

2. Materials and Methods

2.1. Materials

Trypsin from bovine pancreas (pI = 10.1–10.5, M W = 23,800 g·mol–1) was obtained from Sigma-Aldrich. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-(aminopropylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine with different mean molecular weight of PEG chains, M PEG = 2000, 3400, and 5000 g·mol–1 (denoted as DSPE-PEG2000-NH2, DSPE-PEG3400-NH2, and DSPE-PEG5000-NH2, respectively) were obtained from NOF (Tokyo, Japan). Tris-succinimidyl aminotriacetate (TSAT) was obtained from Thermo Scientific. A 25% glutaraldehyde (GA) solution was obtained from Wako Pure Chemicals (Osaka, Japan). N α-Benzoyl-l-arginine-4-nitroanilide hydrochloride (denoted as BAPA) was obtained from Sigma-Aldrich or Peptide Institute, Inc. (Osaka, Japan). Bovine serum albumin (denoted as BSA) was purchased from Sigma-Aldrich. For details concerning the chemicals used, see Table S1 in the Supporting Information. All chemicals were used as received. Water was treated with a water purification system Elix Essential UV3 from Merck.

2.2. Conjugation of Trypsin to Liposomes

Amino-group-bearing liposomes composed of DOPC (50 mg) and DSPE-PEG n -NH2 (molar ratio 95:5) were prepared as follows. Lipids were dissolved in chloroform (4 mL) in a 100 mL round-bottom flask followed by the removal of the solvent using a rotary evaporator. This operation was performed again. Lipid films formed were dried under a high vacuum in the dark for 2 h using a freeze-dryer instrument. The dry lipid film was hydrated with 2.0 mL of a 0.1 M sodium phosphate buffer solution (pH = 7.0) (denoted as PBS), and the lipid suspension was frozen in dry ice/ethanol for 7 min and then incubated at 37 °C for 7 min. This freezing and thawing cycle was carried out 7 times to induce the formation of sufficiently large multilamellar vesicles. To obtain large unilamellar vesicles, the vesicle suspension was passed repetitively through a polycarbonate membrane with nominal pore diameter of 100 nm using an extruder instrument LiposoFast from Avestin. The liposome suspension was analyzed in terms of the DOPC concentration with an enzyme method using a kit LabAssay Phospholipid from Fujifilm Wako, and the total lipid concentration was calculated. Liposomes composed of DOPC and POPE (95:5) were also prepared as described above. The liposome suspension was stored at 4 °C in a 2 mL polypropylene tube until use. To induce a covalent bond between trypsin and liposomes, a liposome suspension, PBS containing trypsin, and dimethyl sulfoxide (DMSO) containing 45 mM TSAT (or a GA solution) were mixed to give the final concentrations of lipids, trypsin, and TSAT (or GA) of 10 mM, 2.5 mg·mL–1, and 3 mM, respectively, in a 2 mL polypropylene tube. A reaction mixture was also prepared at an initial GA concentration of 30 mM. A mixture (1.5 mL) was incubated at 25 °C for 3 h, followed by being loaded onto a sepharose 4B column, collecting each 1.0 mL fraction with PBS as eluent. Twenty fractions collected were analyzed based on trypsin activity with BAPA as the substrate. Earlier eluting turbid fractions corresponded to liposomes conjugated with trypsin. A conjugate formed via TSAT was denoted as liposome-PEG n -TSAT-trypsin and a conjugate via GA as liposome-PEG n -GA-trypsin. Lipid concentration in each conjugate suspension was determined as described above. Liposome-conjugated trypsin was stored at 4 °C in PBS.

2.3. Measurements of Enzyme Activity of Trypsin

Trypsin activity was measured with 1.0 mM BAPA as the chromogenic substrate. − PBS (1080 μL) containing liposome-conjugated or free trypsin was added with 120 μL of DMSO containing 10 mM BAPA in a quartz cuvette with an optical path length l of 1.0 cm. Then, the absorbance at 405 nm (A405) was followed for 3 min at 25 °C as a measure of the formation of p-nitroaniline. Measurements were carried out with a UV-750 spectrophotometer equipped with a temperature controllable cell holder unit EHC-477T from JASCO (Tokyo, Japan). The slope obtained by plotting A405 and the reaction time was taken as the enzyme activity of trypsin. For the relationship between the slope and the concentration of free trypsin, see Figure S1 in the Supporting Information.

2.4. Measurements of Secondary and Tertiary Structures of Trypsin

Circular dichroism (CD) measurements were carried out to estimate the secondary structure content of trypsin. PBS (600 μL) containing liposome-conjugated or free trypsin ([trypsin] = 0.05–0.3 mg·mL–1) was incubated in a 1.5 mL polypropylene tube at 25, 40, or 60 °C for 60 min in an aluminum block temperature controller. Then, the solution was transferred into a quartz cuvette (l = 0.2 cm) for measuring a CD spectrum at 25 °C with an instrument J-1500 equipped with a cell holder unit PTC-510 from JASCO. CD spectra were recorded at the scan rate of 50 nm·min–1 with 0.1 nm intervals. For secondary structure analysis, the CD spectra at 200–250 nm were corrected using corresponding background spectra obtained with PBS only or PBS containing liposome-PEG2000-NH2 or liposome-PEG3400-NH2. Then, the corrected spectra were analyzed using the BeStSel (β Structure Selection) Web server. , The concentration of trypsin in the samples containing conjugates was determined through trypsin activity measurements using BAPA as the substrate and a standard curve made with free trypsin (Figure S1, Supporting Information). Intrinsic fluorescence measurements were carried out to obtain information concerning the tertiary structure of trypsin. PBS containing liposome-conjugated or free trypsin was excited at the wavelength λex of 280 nm, and the emission fluorescence spectrum was measured at 25 °C with a spectrofluorometer FP-8350DS equipped with a cell holder unit EHC-113 from JASCO.

2.5. Measurements of Heat Stability of Trypsin

PBS (typically 700–800 μL) containing liposome-conjugated or free trypsin ([trypsin] = 0.01–0.3 mg·mL–1) was incubated at 40, 50, or 60 °C inside a 1.5 mL polypropylene tube for up to 60 min using the heating aluminum block. Aliquots were periodically withdrawn, followed by being incubated at 25 °C for 3 min using another aluminum block. Then, the enzyme activity was measured with 1.0 mM BAPA as the substrate (see above).

2.6. Measurements of Proteolysis Catalyzed by Trypsin

Proteolytic activity of liposome-conjugated or free trypsin was measured with BSA as the substrate. , PBS (1.0 mL) containing 8.0 mg·mL–1 BSA was incubated in a 1.5 mL polypropylene tube at 95 °C for 15 min using the heating aluminum block to induce the denaturation of BSA. Then, PBS (900 μL) containing denatured BSA (1.0 mg·mL–1) and liposome-conjugated or free trypsin ([trypsin] = 0.01 mg·mL–1) was prepared and incubated at 37 or 50 °C for 60 min. Aliquots (200 μL) were periodically withdrawn, followed by being diluted with PBS (2.8 mL) for the intrinsic fluorescence measurement at λex = 280 nm and 25 °C with the spectrofluorometer. The contribution of the fluorescence of trypsin was negligible due to the high BSA-to-trypsin weight ratio in the assay ([BSA]:[trypsin] = 100:1).

3. Results and Discussion

3.1. Effects of Size of PEG Chain and Cross-linker on the Characteristics of Liposome-Conjugated Trypsin

Covalent conjugation of trypsin molecules to amino-group-bearing liposomes was induced with TSAT or GA as the cross-linker at 25 °C in PBS. Gel permeation chromatography (GPC) was used to separate unbound trypsin from liposome-PEG n -TSAT-trypsin or liposome-PEG n -GA-trypsin. Representative GPC profiles obtained for liposome-PEG2000-GA-trypsin are shown in Figure A (see also Figures S2–S4, Supporting Information for GPC profiles). As shown in Figure A, two eluting peaks based on the trypsin activity were observed with respect to the reaction mixture initially containing trypsin, GA, and liposome incorporated with DSPE-PEG2000-NH2. This result indicates that a colloidal dispersion of liposome-PEG2000-GA-trypsin was eluted in the earlier peak and the unbound enzyme in the later one. On the other hand, GPC of the mixture containing free trypsin and liposomes incorporated with DSPE-PEG2000-NH2 (no GA) gave a single peak corresponding to free trypsin (Figure A). Furthermore, a single peak was also observed for the mixture containing trypsin, GA, and NH2-free liposomes, see also Figure A. Above three GPC profiles consistently indicate that liposome-PEG2000-GA-trypsin was formed through the covalent bond via GA between the amino groups of trypsin and liposomes, and physical adsorption of trypsin to PEG-tethered liposome membranes was negligible. In Figure A, free trypsin being contacted with GA tends to elute earlier than that without GA, suggesting that cross-linked multimers of trypsin were formed via GA. Nevertheless, liposome-PEG2000-GA-trypsin could be effectively separated from free trypsin and its multimers by using GPC. The mean hydrodynamic diameter, D h of trypsin-conjugated liposomes or enzyme-free PEG-tethered liposomes, was determined with the dynamic light scattering method. As shown in Table S2–1, the D h value of liposomes tended to increase through being conjugated with trypsin, which was pronounced for the liposome–trypsin conjugates prepared with GA as cross-linker. Figure B shows effects of the type of cross-linker used and the molecular weight M PEG of the PEG chain of liposomes on the activity-based molar amount of trypsin conjugated per molar amount of lipids, m T. Clearly, the m T value obtained with GA is larger than that with TSAT at any values of M PEG, and the m T value tends to increase as the M PEG value increases, regardless of the type of cross-linker used. Practically no trypsin was conjugated to PEG-free POPE/DOPC liposomes with TSAT. The maximum m T value of 1.41 ± 0.07 mmol-enzyme·(mol-lipids)−1 was obtained for liposome-PEG5000-GA-trypsin. This means that a liposome with diameter of 150 nm was conjugated with trypsin, which exhibited the activity equivalent to 2.6 × 102 free trypsin molecules, and each trypsin was surrounded by 3.7 × 102 lipid molecules on liposome membranes assuming that trypsin was present in the outer lipid layer only, see Table S2–2, Supporting Information.

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Chromatographic purification and characteristics of liposome-conjugated trypsin. (A) GPC elution profile of (i) a reaction mixture initially containing free trypsin, liposomes incorporated with DSPE-PEG2000-NH2, and GA to induce the formation of liposome-PEG2000-GA-trypsin (eluting in fractions 4–6), (ii) a mixture of free trypsin and liposomes incorporated with DSPE-PEG2000-NH2 (no cross-linker), or (iii) a mixture of free trypsin, DOPC liposomes (no NH2 group), and GA. Each mixture was incubated for 3 h at room temperature (≈25 °C) prior to GPC. The volume of each fraction collected was 1.0 mL. The enzyme activity was determined with 1.0 mM BAPA as the substrate in PBS at 25 °C and converted into the apparent trypsin concentration with a separately determined relationship between the activity and concentration of free trypsin (see Figure S1, Supporting Information). (B) The activity-based molar amount of trypsin conjugated per molar amount of lipids is m T as a function of the M PEG value with TSAT or GA as the cross-linker. Data represent the mean ± standard deviation (SD) (n = 2 or 3). With TSAT, practically no trypsin was conjugated to the liposomes (m T ca. 0) at M PEG = 0 (no PEG, just POPE).

Figure shows the CD spectra measured for liposome-PEG2000-GA-trypsin, liposome-PEG3400-GA-trypsin, and free trypsin. The CD spectra obtained for liposome-PEG2000-GA-trypsin and liposome-PEG3400-GA-trypsin are basically the same. The spectra of liposome-conjugated trypsin appear to be different from the spectrum of the free enzyme. However, as shown in Figure S6, Supporting Information, the predicted secondary structure contents estimated using the BeStSel analysis were rather similar to those of the free trypsin and crystal structure (PDB ID 4I8K ): free trypsin, 44% for β-sheet, and no α-helix (root-mean-square deviation, RMSD = 0.097); liposome-PEG2000-GA-trypsin, 29% for β-sheet, and no α-helix (RMSD = 0.45); liposome-PEG3400-GA-trypsin, 32% for β-sheet, and 7% for α-helix (RMSD = 0.22); and trypsin crystal structure (PDB ID: 4I8K), 32% for β-sheet, and 7% for α-helix. These results suggest that liposome-conjugated trypsin retains its β-sheet rich conformation, which is considered critical for maintaining enzymatic activity at the lipid membrane interface, despite potential partial denaturation may occur.

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CD spectrum of free trypsin (gray), liposome-PEG2000-GA-trypsin ([lipid] = 1.6 mM) (blue), or liposome-PEG3400-GA-trypsin ([lipid] = 1.0 mM) (red) in PBS at 25 °C. Mean residue ellipticity [θ] was calculated with the number of amino acid residues of 223 for trypsin and [trypsin] = 0.05 mg·mL–1 for all spectra. For the spectral analysis, see Figures S5–S7, Supporting Information.

For the intrinsic fluorescence spectra of liposome-PEG n -GA-trypsin, see Figure S8, Supporting Information. Slight blue shift in the wavelength of maximum fluorescence emission, λmax was observed with respect to PEG-tethered liposome-conjugated trypsin compared to free enzyme. Furthermore, the maximum fluorescence intensity I max for liposome-conjugated trypsin tended to be larger than that of the free enzyme under the fixed activity-based concentration of the enzyme. Therefore, the specific activity of trypsin may be altered through being conjugated to liposomes.

3.2. Temperature-Dependent Autolysis of Free Trypsin

Figure A shows the remaining activity of free trypsin after being heated at 40 °C in PBS for 60 min. The trypsin concentration used in the heating experiments was varied from 0.05 to 0.3 mg·mL–1. Each heated sample was incubated at 25 °C for 5 min prior to the activity measurement with BAPA as the substrate. Heat-induced significant and irreversible inactivation of free trypsin is seen at all enzyme concentrations examined (Figure A). The remaining activity of trypsin clearly decreases with increasing enzyme concentration at heating, giving the remaining activities of 41 ± 2 and 20 ± 3% at the trypsin concentrations of 0.05 and 0.3 mg·mL–1, respectively. This result indicates that heat inactivation of free trypsin is a reaction involving multiple enzyme molecules and probably involve an enzyme autolysis. Intrinsic fluorescence measurements were carried out with respect to PBS containing free trypsin being heated at 40 °C for 60 min at various enzyme concentrations; see Figure S9, Supporting Information. The maximum fluorescence intensity observed after heating, I max,60 significantly decreased compared to the initial maximum intensity, I max,0. Furthermore, a clear red shift in the λmax value was observed after heating. The Δλmax value, which was calculated as Δλmax = λmax,60 – λmax,0, where λmax,0 and λmax,60 represent the λmax values without and with heating, respectively. The values of Δλmax and I max,60·I max,0 –1 are plotted as a function of enzyme concentration, see Figure B. The I max,60·I max,0 –1 value can be a quantitative measure of the heating-induced quenching of the fluorescence, while the Δλmax value corresponds to the degree of heat-induced solvent exposure of the aromatic amino acid residues within the enzyme. As seen in Figure B, the value of Δλmax clearly increases with increasing enzyme concentration, showing Δλmax = 13 ± 1 nm at [trypsin] = 0.3 mg·mL–1. Furthermore, the value of I max,60·I max,0 –1 tends to decrease with increasing enzyme concentration, meaning that fluorescence quenching was pronounced at high enzyme concentrations. These results consistently show that the tertiary structure of free trypsin was significantly lost by the enzyme autolysis in the concentration-dependent manner. Figure C shows the CD spectra of PBS containing 0.25 mg·mL–1 free trypsin being heated at 25, 40, or 50 °C for 60 min. Clearly, the secondary structure of free trypsin at 25 °C is destroyed irreversibly at 40 °C. The structure of free trypsin at 40 °C is comparable to that observed at 50 °C (Figure C), see also Figures S10 and S11, Supporting Information for the spectrum at [trypsin] = 0.05 mg·mL–1 and the relevant spectral analysis, respectively. In contrast to free trypsin, the intrinsic fluorescence spectrum of liposome-PEG2000-GA-trypsin in PBS was not significantly changed by being incubated at 40 °C for 60 min (Δλmax = −0.5 ± 0.5 nm, I max,60/I max,0 = 92 ± 1%, n = 3), see Figure S12, Supporting Information for the spectra. Furthermore, 110 ± 11% of the initial activity of liposome-PEG2000-GA-trypsin was observed after the above heat treatment.

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Heat stability and conformational structure of free trypsin at different enzyme concentrations. (A) Remaining activity of free trypsin (0.05–0.3 mg·mL–1) after being heated at 40 °C in PBS for 60 min. (B) The difference in the wavelength of the maximum fluorescence intensity, Δλmax of a free trypsin solution before and after being heated at 40 °C for 60 min, is a function of the concentration of free trypsin. The maximum fluorescence intensity of a free trypsin solution with heating under the above conditions, I max,60 relative to that without heating, I max,0 is also plotted as a function of the enzyme concentration. See Figure S9 in the Supporting Information for the corresponding fluorescence spectra. (C) Mean residue ellipticity [θ] of a free trypsin solution ([trypsin] = 0.25 mg·mL–1) incubated at 25, 40, or 50 °C for 60 min in PBS.

3.3. Effects of Lipid Membranes on Heat Stability of Liposome-Conjugated Trypsin

In the next series of experiments, stability of enzyme activity was compared at 50 or 60 °C among the following three catalyst systems; (i) liposome-PEG2000-TSAT-trypsin, (ii) free trypsin, and (iii) a mixture of free trypsin and free liposomes. In Figure A (T = 50 °C, [trypsin] = 0.05 mg·mL–1), a progressive enzyme deactivation is seen over time for free trypsin and a free trypsin/free liposome mixture. For liposome-PEG2000-TSAT-trypsin, on the other hand, its heat stability was clearly higher than in the case of the other two catalyst systems. At 60 °C and [trypsin] = 0.01 mg·mL–1, liposome-PEG2000-TSAT-trypsin also showed the highest heat stability (Figure B), although the rate of enzyme deactivation is accelerated for all catalyst systems compared to the results obtained at 50 °C (Figure A). At 60 °C, a slight stabilization effect of free liposomes was seen on the enzyme activity. These results demonstrate that trypsin became significantly heat stable through being conjugated to PEG-tethered lipid membranes via a chemical linkage, whereas liposome membranes had only a weak stabilizing effect on the coexisting free trypsin molecules under the fixed overall concentrations of trypsin and lipids.

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Remaining activity of liposome-PEG2000-TSAT-trypsin, free trypsin, and the mixture of free trypsin and free liposomes incorporated with 5 mol % DSPE-PEG2000-NH2 in PBS at 50 °C (A) or 60 °C (B). The trypsin concentration was fixed at 0.05 mg·mL–1 (A) or 0.01 mg·mL–1 (B). The lipid concentration of the liposome-containing sample was 6.0 mM (A) or 4.0 mM (B). Data represent mean ± SD. For each data and the number of independent experiments performed for each catalyst, see Tables S3–S5 in the Supporting Information.

Figure depicts the stability of various liposome-conjugated trypsin at 50 °C at [trypsin] = 0.01 mg·mL–1. For comparison, the heat stability of free trypsin at the same enzyme concentration is shown in Figure A. Free trypsin activity is confirmed to be unstable, showing the remaining activity of 21 ± 3% at 60 min. On the other hand, liposome-PEG3400-TSAT-trypsin and liposome-PEG5000-TSAT-trypsin showed significantly higher remaining activity of 60 ± 8% and 93%, respectively, at 60 min heating (Figure A). Liposome-PEG n -GA-trypsin (n = 0, 2000, 3400, or 5000) also consistently showed significantly higher heat stability than free trypsin, see Figure B. These results demonstrate that the heat stability of trypsin could be significantly improved through being conjugated to liposomes possessing any size of PEG chain and with TSAT or GA as the cross-linker, see also Figure S13, Supporting Information.

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Heat stability of liposome-conjugated or free trypsin at 50 °C. (A) Time-dependent remaining activity of liposome-PEG n -TSAT-trypsin or free trypsin in PBS at 50 °C ([trypsin] = 0.01 mg·mL–1). (B) Stability of liposome-PEG n -GA-trypsin in PBS was examined at 50 °C under otherwise the same condition as described for panel (A). Data represent mean ± SD. For each data and the number of independent experiments performed for each catalyst, see Tables S3, S6–S11 in the Supporting Information.

3.4. Controlled Proteolysis Reactions Catalyzed by Liposome-Conjugated Trypsin

Proteolytic activity of liposome-conjugated or free trypsin was examined with BSA as the proteinaceous substrate at 37 °C for 240 min in PBS. The degree of trypsin-catalyzed hydrolysis was evaluated by following the intrinsic fluorescence intensity of BSA in a reaction mixture. Note that the contribution of trypsin to the overall fluorescence spectrum was negligible ([BSA]:[trypsin] = 100:1 in weight ratio). Figure A shows the time-dependent fluorescence spectrum obtained with respect to the mixture initially containing 1.0 mg·mL–1 BSA being pretreated at 95 °C for 15 min followed by the addition of free trypsin at 25 °C and 0.01 mg·mL–1 for performing the catalytic proteolysis at 37 °C. In the figure, the maximum fluorescence intensity decreases as the reaction time elapsed. This result indicates that BSA was hydrolyzed by the catalytic action of free trypsin, which led to alterations in the tertiary structure of BSA. Proteolytic activity of free trypsin to BSA was also observed based on the measurement of aromatic amino acid residues released in the reaction mixture; see Figure S14 in the Supporting Information. In Figure B, the time-dependent fluorescence spectrum is shown for the mixture initially containing BSA (1.0 mg·mL–1) and liposome-PEG2000-GA-trypsin ([trypsin] = 0.01 mg·mL–1). The maximum fluorescence intensity is seen to decrease over time, indicating that the catalytic digestion of BSA was caused by liposome-conjugated trypsin. The data obtained with the mixture of BSA and enzyme-free liposomes ([lipids] = 2.0 mM) are shown in Figure C. Little change in the fluorescence intensity was observed throughout the incubation time of 240 min, clearly demonstrating that intact lipid membranes were catalytically silent toward BSA. Therefore, the result shown in Figure B means that liposome-conjugated trypsin preserved its catalytic activity to a macromolecular substrate (BSA) present in bulk solution. Hydrolysis of BSA was also examined with various liposome-conjugated trypsin as a potential catalyst at a fixed trypsin concentration of 0.01 mg·mL–1, see Figures S15 and S16 in the Supporting Information. To compare the performance of various reaction systems, relative fluorescence intensity of a reaction mixture, I t·I 0 –1, at the emission wavelength, λem = 325 nm, was calculated as the intensity after being incubated for a certain reaction time, I t, divided by the initial intensity, I 0. The time-dependent I t·I 0 –1 value calculated is shown in Figure . In Figure A, the I t·I 0 –1 value obtained with free trypsin decreases to 63 ± 2% at t = 240 min, while it decreases to 99 ± 2% with enzyme-free liposomes. Liposome-PEG n -TSAT-trypsin showed significantly lower proteolytic activity to BSA compared to free trypsin, see Figure B. Practically no proteolytic activity was seen in the case of liposome-PEG2000-TSAT-trypsin and liposome-PEG5000-TSAT-trypsin. Catalytic performance of liposome-PEG n -GA-trypsin including liposome-GA-trypsin (n = 0) is shown in Figure C. Liposome-PEG n -GA-trypsin clearly exhibited proteolytic activity which is consistently lower than the activity of free trypsin (Figure A). The results shown in Figure B and C demonstrate that liposomes offer a reaction environment that moderately or almost completely inhibits the proteolytic activity of surface-conjugated trypsin. It should also be noted that liposome-GA-trypsin (no PEG moiety) exhibited higher proteolysis activity than liposome-PEG n -GA-trypsin (n = 2000 or 5000) (Figure C). The reactivity of each catalyst toward BAPA or heat-inactivated BSA is schematically illustrated in Figure . It could be that the low activity of liposome-bound trypsin toward macromolecular substrates is due to steric hindrance caused by lipid membranes. The interaction of the active site of liposome-bound trypsin with a large substrate (BSA) may be sterically hindered. For a small molecule, like BAPA, access to the active site of trypsin in the liposomal environment may be possible more easily. Furthermore, partially hydrophobic BSA may also bind to the liposomes away from the trypsin molecules conjugated to PEG-tethered lipid membranes, preventing interaction with trypsin. For the results concerning the proteolysis reactions under different conditions and the stability of free trypsin with BSA, see Figures S17 and S18 in the Supporting Information, respectively. The above results indicate that the factors, which potentially affect the proteolysis activity of liposome-conjugated trypsin, are the density of trypsin on the liposome surface (trypsin/lipid ratio in a reaction system) and the conformation of surface-conjugated trypsin. In other words, trypsin conjugation to a PEG-tethered liposome offers a wide range of opportunity to modulate the proteolysis activity of trypsin through appropriately selecting the size of PEG chains and the type of chemical linkage between the enzyme and liposomes.

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Proteolysis reactions catalyzed by free or liposome-conjugated trypsin with 1.0 mg·mL–1 BSA as the substrate at 37 °C in PBS. Intrinsic fluorescence spectra of PBS with (A) free trypsin ([trypsin] = 0.01 mg·mL–1), (B) liposome-PEG2000-GA-trypsin ([trypsin] = 0.01 mg·mL–1), or (C) enzyme-free liposomes ([lipid] = 2.0 mM). Each mixture was incubated in PBS (900 μL) for 240 min. Aliquots (200 μL) of a mixture were periodically withdrawn followed by being diluted with 2800 μL of PBS for the fluorescence measurement at λex = 280 nm and 25 °C.

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Proteolysis of 1.0 mg·mL–1 BSA at 37 °C catalyzed by free or various types of liposome-conjugated trypsin. Time courses of the relative fluorescence intensity I t·I 0 –1, where I t and I 0 represent the intensity at λem = 325 nm at the reaction time t and initial state, respectively, for the mixture of BSA and (A) enzyme-free liposomes ([lipid] = 2.0 mM) or free trypsin (0.01 mg·mL–1), (B) liposome-PEG n -TSAT-trypsin ([trypsin] = 0.01 mg·mL–1), or (C) liposome-PEG n -GA-trypsin ([trypsin] = 0.01 mg·mL–1). Data represent the mean ± SD (n = 3).

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Schematic illustrations of the reactions catalyzed by free or liposome-conjugated trypsin with BAPA (A) or heat-inactivated BSA (B) as a substrate. (A) Small BAPA molecules are considered to be readily accessible to the active site of free trypsin and the enzyme being conjugated to liposomes. (B-i) Free trypsin efficiently catalyzes the digestion of BSA yielding a degraded protein. (B-ii) Liposome-GA-trypsin (trypsin conjugated via GA to a liposome without PEG) and (B-iii) liposome-PEG n -GA-trypsin also catalyze the proteolytic reaction. The proteolytic activity of free trypsin is higher than that of liposome-conjugated trypsin, and liposome-GA-trypsin shows higher activity than liposome-PEG n -GA-trypsin. (B-iv) Liposome-PEG n -TSAT-trypsin exhibits little or significantly low activity toward BSA. Partially hydrophobic BSA molecules may interact with lipid membranes, which can affect the accessibility of BSA to trypsin on the surface of liposomes without or with PEG moiety.

4. Conclusions

Liposome-PEG n -(GA or TSAT)-trypsin was prepared by conjugating trypsin to preformed PEG-tethered liposomes via GA or TSAT as the cross-linker. Conjugation reaction with GA gave liposomes with significantly higher active enzyme density than with TSAT. Heat-induced deactivation of free trypsin was significant at 40–60 °C because of the involvement of autolysis. On the other hand, homogeneously dispersed liposomes carrying surface-conjugated trypsin were remarkably heat stable at 40–60 °C in their enzyme activity, clearly demonstrating that trypsin–liposome conjugation resulted in effective inhibition of autolysis and thermal denaturation of the enzyme. Furthermore, the liposome-conjugated trypsin exhibited proteolytic activity toward heat-inactivated BSA. Compared to free trypsin, liposome-bound trypsin was less efficient in catalyzing the degradation of BSA. For the liposome-bound trypsin, significant BSA degradation was only observed for liposome-PEG n -GA-trypsin, with highest activity for a PEG-free conjugate (n = 0). The low activity of liposome-bound trypsin toward this macromolecular substrate may be due to restrictions of the access of BSA to liposome-bound trypsin. The challenge remaining is to clarify the combined effects of polymer chains and enzyme density on the proteolysis activity of trypsin conjugated to liposomes. In summary, liposome-PEG n -GA-trypsin was useful as a highly biocompatible and self-dispersible colloidal biocatalyst with controllable proteolytic activity. The present liposome-PEG n -GA-trypsin enables catalytic proteolysis reactions to be carried out at temperatures higher than those under conventional conditions.

Supplementary Material

ao5c06073_si_001.pdf (479.6KB, pdf)

Acknowledgments

The authors thank the financial support from the JSPS KAKENHI (grant nos. JP 23K26470 and JP 22K18923).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c06073.

  • Chemicals used; activity-concentration relationship for free trypsin; GPC profiles for the purification of liposome-conjugated trypsin; size and characteristics of trypsin-conjugated liposomes; analysis of CD spectra; intrinsic fluorescence spectrum of liposome-conjugated trypsin; intrinsic fluorescence spectrum of free trypsin; temperature-dependent CD spectrum of free trypsin; tertiary structure of liposome-conjugated trypsin at 40 °C; heat stability of liposome-conjugated or free trypsin; proteolysis reactions catalyzed by liposome-conjugated trypsin (PDF)

M.W. designed and performed experiments concerning the preparation, characteristics, and proteolysis activity of liposome-conjugated trypsin and analyzed data. N.Y. conducted CD measurements with respect to liposome-conjugated and free trypsin, calculated the secondary structure of trypsin based on the CD spectra, and analyzed the structural characteristics of trypsin. N.Y. also carried out CD measurements for enzyme-free PEG-tethered liposomes. M.Y. conducted the research concerning the preparation, thermal stability, and catalytic activity of liposome-conjugated and free trypsin and analyzed data. This article was written through contributions of all authors.

Japan Society of Promotion of Science (JSPS) KAKENHI Grant Numbers JP 23K26470 and JP 22K18923.

The authors declare no competing financial interest.

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Supplementary Materials

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