Article pubs.acs.org/Langmuir
Selective Oxidation of D‑Amino Acids Catalyzed by Oligolamellar Liposomes Intercalated with D‑Amino Acid Oxidase Makoto Yoshimoto,* Masakazu Okamoto, Kouta Ujihashi, and Takayuki Okita Department of Applied Molecular Bioscience, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan S Supporting Information *
ABSTRACT: D-Amino acid oxidase (DAO) is structurally unstable and exhibits broad specificity to D-amino acids. In this work, we fabricated a stable liposomal DAO system with high apparent substrate specificity. Permeability of the membrane composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) was highly selective between the D-forms of alanine (Ala) and serine (Ser). The permeability coefficient of D-Ala and D-Ser at 25 °C was 3.59 and 0.27 pm/s, respectively, as determined with the dialysis method. On the other hand, the chiral environment of POPC membrane showed no clear selectivity between the enantiomers of Ala or Ser. POPC liposomes encapsulating DAO from porcine kidney selectively catalyzed the oxidation of hydrophobic D-phenylalanine (D-Phe) over D-Ala and D-Ser because of their intrinsic membrane permeability. As a different type of liposomal DAO, the enzyme molecules were conjugated to the surface of activated lipids-bearing liposomes. The activity of liposome-conjugated DAO showed significantly higher stability at 50 °C than free DAO at low enzyme concentrations ranging from 2.5 to 10 mg/L. Then, the DAO-conjugated liposomes were coated with POPC bilayers to give the oligolamellar structure intercalated with the DAO molecules. The additional bilayers allowed to induce the permeability resistance-based substrate specificity and strengthened the stabilizing effect on the DAO activity. The oligolamellar liposomes fabricated can be a colloidal platform for integrating the functions of lipid membrane to stabilize DAO and to modulate its substrate specificity.
1. INTRODUCTION Major constituents of biopolymers in living systems are homochiral compounds such as L-α-amino acids and D-sugars.1 Although D-amino acids are minor constituents, they are recognized to be relevant to various biological phenomena such as aging and diseases of mammals.2,3 For instance, the accumulation level of D-serine in the human brain is related to its disorders.4−6 Furthermore, the racemization of naturally occurring L-amino acids is induced during food processing, which alters digestibility and nutritional characteristics of food proteins.2,3 Therefore, D-amino acids can be one of the key compounds of diseases in the pharmaceutical industry and also indicators of the quality of foods and beverages. In this context, selective quantification of low concentrations of D-amino acids is of significance and relevant methods for the quantification need to be developed.7 D-Amino acid oxidase (DAO, EC 1.4.3.3) is an enzyme that catalyzes the oxidative deamination of D-amino acids with molecular oxygen producing α-keto acids, hydrogen peroxide, and ammonia.8 The DAO reaction is advantageous to exclusively oxidize D-forms of amino acids in their racemic mixtures. The DAO reaction-based devices were reported for the sensitive detection of D-amino acids for biochemical and biomedical purposes.9−14 The DAO molecules immobilized in solid supports such as polymer-modified electrodes9 and carbon nanotubes11 can be practical sensor systems particularly © 2014 American Chemical Society
because of their reusability. On the other hand, several features of DAO are generally disadvantageous in practical applications. The DAO activity is extraordinary unstable because of structural fragility15 and dissociation of the enzyme-incorporated flavin adenine dinucleotide (FAD) molecules.16 Furthermore, DAO possesses a board spectrum of specificity to Damino acids,17,18 which is unfavorable for the selective oxidation of a particular D-amino acid. Therefore, the reaction system that can regulate both stability and substrate specificity of DAO would be beneficial for practical applications of the enzyme reaction. Liposome membranes offer a unique colloidal platform for chemical reactions19,20 and stabilization of the enzyme molecules.21 The membrane interface can stabilize the partially denatured enzyme molecules through interfering with intermolecular interaction among the conformationally changed enzyme molecules.22 We recently reported that the DAO activity was stabilized at a high temperature through being coupled to liposomes.23 Probably, the quaternary structure of DAO was stabilized through the covalent bonding as well as the enhanced hydrophobic interaction with the host membranes. We also reported an approach to prepare oligolamellar Received: February 26, 2014 Revised: April 19, 2014 Published: May 12, 2014 6180
dx.doi.org/10.1021/la500786m | Langmuir 2014, 30, 6180−6186
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used. The membrane possessed a flat width of 16 mm and the approximate molecular weight cutoff of 20 kDa. The tubular membrane was filled with 1.0 mL of the amino acid-containing liposome suspension ([POPC] = 10 mM) using closures at both end of the tube. Then, the dialysis was carried out against 100 mL of the phosphate buffer solution containing 50 mM NaCl with gentle stirring. Temperature of the dialysis system was kept at 25 ± 0.3 °C in a water bath instrument TBL310GA from Advantec Toyo (Tokyo, Japan). Aliquots (1.5 mL) were withdrawn from the buffer solution, and the concentration Ct of amino acid was determined by the fluorescamine method28 as follows. The sample (1125 μL) was mixed with 375 μL of the acetone solution containing 0.015 wt % fluorescamine followed by vortexing. Then, fluorescence intensity of the solution was measured at the excitation and emission wavelengths of 400 and 475 nm, respectively with a spectrofluorometer instrument FP-750 from JASCO (Tokyo, Japan). The concentration of each amino acid was determined with the respective relationship between the intensity and its concentration. The concentrations of amino acids at permeation equilibrium C∞ were determined by releasing the liposome suspension to outside the dialysis tube and solubilizing the liposomes with 2.0 mM Triton X-100 at the fluorescence measurement. The fractional amino acid (Raa) released at any time t was calculated as Raa = Ct/C∞. Preparation of POPC Liposomes Encapsulating DAO and Catalase. A dry POPC (50 mg) film was prepared as described above. The lipid film was hydrated with 2.0 mL of a 50 mM phosphate buffer solution of pH 9.0 containing 1.0 mg/mL DAO and 5.0 mg/mL catalase. The freezing and thawing treatments and the sizing of liposomes were performed with the same procedures as described above. Free enzyme molecules, which were not encapsulated in liposomes, were separated from the liposomes encapsulating the enzymes using the GPC with a salt-free phosphate buffer solution as eluent. Measurements of Enzyme Activity. The enzyme activity of DAO was measured with 10 mM D-Ala as substrate. The increase in concentration of H2O2, which is one of the products of DAO-catalyzed reaction, was continuously followed at 25 °C based on the horse radish peroxidase (HRP)-catalyzed oxidation of o-dianisidine with the H2O2.29 The oxidized o-dianisidine was detected based on the absorbance at 460 nm using a spectrophotometer (V-550, JASCO, Japan) with the molar extinction coefficient30 of 11 300 M−1 cm−1. The intrinsic activity of DAO was measured in the presence of 2.0 mM Triton X-100 for the solubilization of liposome membranes. One unit of DAO is defined as the amount of enzyme that catalyzes the oxidation of 1.0 μmol of D-Ala per minute at 25 °C in the phosphate buffer solution. Oxidation of Amino Acids Catalyzed by Liposomal Systems. Catalytic oxidation of D-Ala, D-Ser, or D-Phe was performed at 25 °C in the phosphate buffer solution suspending the liposomes encapsulating DAO and catalase at the lipid concentration of 5.0 mM. The reaction was initiated by the addition of a D-amino acid solution to give its initial concentration of 2.5 mM. Aliquots (50 μL) were withdrawn periodically to quantify the residual concentration of amino acid with the fluorescamine method. For comparison, the reaction was performed with the enzyme-containing liposomes in the presence of 22.6 mM Triton X-100 to give the mixture of lipid/Triton X-100 micelles and the free DAO molecules. Preparation of Unilamellar Liposomes Conjugated with DAO (UV-DAO) and Oligolamellar Liposomes Intercalated with DAO (OV-DAO). The liposomes composed of POPC, DOPENHS, and cholesterol (55:15:30 in molar ratio) were prepared according to the procedure reported previously for the liposomes with different composition.23 In this work, DOPE-NHS was employed, whereas in the previous work NGPE (1,2-dioleoyl-sn-glycero-3phosphoethanolamine-N-(glutaryl)) was used.21,23 The coupling reaction between the amino group of DAO and the liposome membrane-incorporated DOPE-NHS was initiated by mixing 1.0 mL of the liposome suspension containing 50 mM EDC and 1.0 mL of a 50 mM borate/100 mM NaCl buffer solution of pH 7.4 containing 1.0 mg/mL DAO. The mixture was incubated at 25 °C for 3 h to induce the formation of lipid−DAO coupling. The UV-DAO was separated
liposomes where the DAO molecules were stably confined between two lamellae,23 although reactivity of the liposomal enzyme was not clarified. In view of enzyme reactions, the mass transfer resistance through the highly ordered lipid membranes is a factor that can modulate the substrate specificity of enzyme as previously demonstrated for several proteinases.24,25 Therefore, the liposomal system encapsulating DAO can be an attractive candidate for the selective oxidation of D-amino acids.26 In this work, we characterized the apparent substrate specificity of the DAO reaction in the liposomal system through analyzing the membrane permeability of D-amino acids and performing the oxidation of the amino acids catalyzed by the liposomes encapsulating DAO. We also examined an efficient method for the preparation of DAO-conjugated liposomes and their stability was characterized. Then, to integrate the characteristics of the above two liposomal systems, we fabricated oligolamellar liposomes intercalated with the DAO molecules and the apparent specificity of the enzyme activity toward D-amino acids was clarified.
2. EXPERIMENTAL SECTION Materials. D-Amino acid oxidase (DAO) from porcine kidney was obtained from Sigma-Aldrich (St. Louis, MO). 1-Palmitoyl-2-oleoyl-snglycero-3-phosphocholine (POPC, >99%, commercial name: COATSOME MC-6081) and N-(succinimidyloxyglutaryl)-L-α-phosphatidylethanolamine, dioleoyl (DOPE-NHS, ≥85%, COATSOME FE8181SU5), were purchased from NOF (Tokyo, Japan). D-Alanine (D-Ala, ≥98%), L-alanine (L-Ala, ≥98%), fluorescamine, and Triton X100 were purchased from Sigma-Aldrich. D-Serine (D-Ser, >99%), Lserine (L-Ser, >99%), D-phenylalanine (D-Phe, >99%), bovine liver catalase (EC 1.11.1.6), acetone (infinity pure), and cholesterol were purchased from Wako Pure Chemical Industries (Osaka, Japan). 1Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) was purchased from Dojindo Laboratories (Kumamoto, Japan). All chemicals were used as received. Water was sterilized and deionized to give the minimum resistance of 15 MΩ·cm by the instrument Elix 3UV from Millipore (Billerica, MA). Preparation of POPC Liposomes Encapsulating Chiral Amino Acids. Seventy-five milligrams of POPC powder was dissolved in 4.0 mL of chloroform, and the solvent was removed by evaporation under reduced pressure. The POPC was redissolved in 4.0 mL of diethyl ether, and the solvent was removed by the evaporation. This procedure was performed twice. The thin POPC film formed was subjected to the high vacuum (
