Bioconjugate Chem. 2004, 15, 1055−1061
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Mechanism for High Stability of Liposomal Glucose Oxidase to Inhibitor Hydrogen Peroxide Produced in Prolonged Glucose Oxidation Makoto Yoshimoto,† Yuya Miyazaki,† Mitsunobu Sato,† Kimitoshi Fukunaga,† Ryoichi Kuboi,‡ and Katsumi Nakao*,† Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan, and Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka 560-8531, Japan. Received April 13, 2004; Revised Manuscript Received July 13, 2004
Glucose oxidase (GO) was encapsulated in the liposomes composed of POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine) to increase the enzyme stability through its decreased inhibition because of hydrogen peroxide (H2O2) produced in the glucose oxidation. The GO-containing liposomes (GOLs) were completely free of the inhibition even in the complete conversion of 10 mM glucose at 25 °C because the H2O2 concentration was kept negligibly low both outside and inside liposomes throughout the reaction. It was interestingly revealed that the H2O2 produced was decomposed not only by a slight amount of catalase originally contained in the commercially available GO but also by the lipid membranes of GOL. As compared to the GOL-catalyzed reaction, the free GO-catalyzed reaction more highly accumulated H2O2 because of the more rapid glucose conversion despite containing free catalase, leading to the completely inhibited GO before reaching a sufficient glucose conversion. This suggested that only the liposomal catalase could continue to catalyze the H2O2 decomposition. The effect of the glucose oxidation rate, i.e., the H2O2 production rate on the liposomal GO inhibition, was also examined employing the various GOLs with different permeabilities to glucose present in their external phase. It was concluded that the liposomal GO free of the inhibition could be obtained when the GOL-catalyzed H2O2 formation rate was limited by such a suitable lipid bilayer as POPC membrane so that the rate was well-balanced with the sum of the above two H2O2 decomposition rates. The highly stable GOL obtained in the present paper was shown to be a useful biocatalyst for the prolonged glucose oxidation.
INTRODUCTION
Glucose oxidase (GO)-catalyzed glucose oxidation producing gluconic acid and hydrogen peroxide (H2O2) has been utilized for glucose quantification (1, 2), sterilization (3, 4), textile bleaching (5), removal of oxygen from foods (6) and a large-scale production of gluconic acid (7). Immobilization of GO in the various solid supports has been studied for increasing physical stability and reusability of the enzyme (1, 2, 5-7). However, it is usually difficult to obtain the stable glucose oxidase free of the product (H2O2) inhibition to the enzyme in a prolonged glucose oxidation (8). Even though catalase is added to the reaction solution for catalyzing the decomposition of H2O2, the inhibitory effect to catalase also needs to be taken into consideration (9). Therefore, the key to develop a durable and biocompatible system for the prolonged glucose oxidation is considered to be regulating the behavior of H2O2 produced throughout the reaction period with such a biological mean as maintaining the initial activities of both enzymes. We have studied liposomes (phospholipids vesicles) in modulating the reactivity and selectivity of the liposomal enzyme to the externally added substrates (10, 11), refolding denatured enzyme in the presence of liposomes * To whom correspondence should be addressed. Telephone: +81-836-85-9271. Fax: +81-836-85-9201. E-mail: knakao@ yamaguchi-u.ac.jp. † Yamaguchi University. ‡ Osaka University.
(12), and using the liposomal enzyme in a gas-liquid twophase flow in a bubble column (13). These contributions are for developing the nanoscaled biofunctional materials applicable to bioprocesses. These unique features of liposomes are mainly attributed to the selective permeability of the membranes to substrate molecules (10, 11, 13) and the electrostatic and hydrophobic interactions between partially unfolded enzyme and lipid bilayer membranes (12, 14). On the basis of such functions of liposomes as above, we recommended the liposomal GO as a stable biocatalyst for the air oxidation of glucose in an external loop airlift bioreactor. In particular, it is practically important that the commercially available GO encapsulated in liposomes can catalyze a prolonged air oxidation of glucose with remarkably higher stability than free GO (13). This indicates that the enzyme inhibition because of H2O2 produced in the oxidation is effectively suppressed in the liposomal system. However, the mechanism for this phenomenon has so far remained unclear. In the present paper, a mechanism for an increase in the stability of liposomal GO is examined on the basis of the quantitative determination of the H2O2 produced in the glucose oxidations catalyzed by the different types of liposomal GO under the various conditions. The results obtained clearly show that an appropriate liposomal encapsulation of GO can give an extraordinary stable system for the simultaneous glucose oxidation and H2O2 decomposition with negligible formation of the inhibited enzymes.
10.1021/bc049909n CCC: $27.50 © 2004 American Chemical Society Published on Web 08/11/2004
1056 Bioconjugate Chem., Vol. 15, No. 5, 2004 EXPERIMENTAL PROCEDURES
Materials. GO from Aspergius niger (EC 1.1.3.4, 429 units/mg, Mr = 180 000, pI ) 4.44) was purchased from Sigma (St. Louis, MO). The GO from Sigma was nominally low in catalase, with the catalase content of less than 0.1 units/mg protein according to the specification. GO (147 units/mg) was also obtained from Toyobo Co. Ltd. (Osaka, Japan). POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) with purity more than 99% was from Avanti Polar Lipids Inc. (Alabaster, AL). β-DGlucose, H2O2 solution (35.1%), 3-AT (3-amino-1,2,4triazole), DLPC (L-R-phosphatidylcholine, dilauroyl), DMPE (L-R-phosphatidylcholine, dimyristoyl), and sodium cholate were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of GO-Containing Liposomes (GOL). The GOL was prepared by the extrusion technique as reported previously (10), essentially based on the method of Mayer et al. (15) as follows. The lipid (30-100 mg) was at first solubilized in chloroform in a round-bottom flask followed by removal of the solvent by using a rotary evaporator. This procedure was repeated twice using diethyl ether as the second solvent. The lipid film obtained was dried under reduced pressure (
