Analysis of Adsorption Function of Polyhydroxybutyrate Depolymerase

Biomacromolecules 2001, 2, 25-28

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Analysis of Adsorption Function of Polyhydroxybutyrate Depolymerase from Alcaligenes faecalis T1 by Using a Quartz Crystal Microbalance Koichi Yamashita,* Yoshihiro Aoyagi, Hideki Abe, and Yoshiharu Doi Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan Received August 14, 2000; Revised Manuscript Received November 10, 2000

Enzymatic adsorption and degradation of three types of aliphatic polyester films and two types of polyolefin films by the extracellular PHB depolymerase from Alcaligenes faecalis T1 have been studied by using a quartz crystal microbalance (QCM) technique. Hydrolysis of poly[(R)-3-hydroxybutyrate] was quantitatively followed by the QCM technique. Adsorption of the enzymes to films was also quantitatively detected by the QCM. Kinetic study on enzymatic adsorption suggests that the PHB depolymerase binds to the substrates not only by hydrophobic interaction but also by specific interaction between the ester bonds of polyesters and the binding domain of the enzyme. The results show that the QCM technique is a sensitive tool to study enzymatic degradation kinetics of biodegradable polyesters. Polyhydroxyalkanoates (PHAs) are synthesized and accumulated within cells in a wide variety of bacteria as a carbon- and energy-storage material.1-3 Since these microbial PHA polymers are biodegradable thermoplastics, intensive studies have been carried out toward practical use. PHAs are degraded by a number of microorganisms such as bacteria and fungi in various environments. These microorganisms excrete polyhydroxybutyrate (PHB) depolymerases to hydrolyze the solid-state PHAs into water-soluble oligomers and monomers. Several extracellular PHB depolymerases have been purified from bacterial strains such as Alcaligenes faecalis,4,5 Pseudomonas lemoignei,6-9 Pseudomonas stutzeri,10,11 Comamonas testosteroni,12,13 and Comamonas acidoVorans.14 Characterization of the structural genes has revealed that PHB depolymerases are composed of two domains and a linker region.15,16 One of the domains is a substrate-binding domain by which the enzymes bind to solid PHAs. Another domain is a catalytic domain that has a lipase box and hydrolyzes the ester bonds of PHA chains. The linker region flexibly connects these two domains. The functions of the catalytic domain and the binding domain should be revealed to understand the properties of the PHB depolymerases. From kinetic analysis, it has been proposed that hydrolysis of water-insoluble PHAs proceeds by a two-step reaction, namely, adsorption of the depolymerase to the surface of the polymer and subsequent hydrolysis.17 Enzymatic hydrolysis of water-soluble 3-hydroxybutylate (3HB) oligomers with the wild-type PHB depolymerase from Alcaligenes faecalis T1 gave valuable information concerning the catalytic function of the active site.5,18 Bachmann and Seebach proposed that (i) the PHB depolymerase from A. faecalis T1 is an endo esterase, (ii) its active site has four * To whom correspondence may be addressed. Phone: +81-48(467)8000. Fax: +81-48(462)4668. E-mail: [email protected].

subsites, and (iii) the central two subsites recognize two (R)3HB units.18 In a previous study, we studied enzymatic hydrolysis of water-soluble 3HB oligomers with two deletion mutants lacking the substrate-binding domain and linker region of PHB depolymerase from Pseudomonas stutzeri.19 The results suggested that the PHB depolymerase from P. stutzeri recognized four monomeric units or three ester bonds in an active site. On the basis of product analysis, we have concluded that the substrate recognition in the active site of the depolymerase from P. stutzeri is different in specificities from that of the depolymerase from A. faecalis T1. The binding characteristic of a PHB depolymerase was studied by using glutathione-S-transferase (GST) fusion proteins with substrate-binding domain of four bacterial PHB depolymerases, A. faecalis, P. stutzeri, C. testosteroni, and C. acidoVorans.20,21 We have suggested that recognition of water-insoluble PHAs by the substrate-binding domain includes not only a simple hydrophobic interaction but also chemical interaction between the several amino acids in the binding domain and the surface of polyesters.21 However, the mechanism of the enzyme binding is still unclear because of difficulty in monitoring enzyme adsorption directly in real time. Recently, the quartz crystal microbalance (QCM) has been shown to be a sensitive tool for the kinetic study of protein adsorption.22-29 The QCM is based on the piezoelectric nature of a quartz crystal. The change of mass on the QCM is electrically detected as the shift in resonant frequency with sensitivity in the ng/cm2 regime.30 In this study, we have developed the QCM setup to monitor the reaction of the PHB depolymerases with polymer films. Enzyme adsorption and enzymatic degradation of PHA films would be detected as a negative and positive frequency shifts, respectively. In this Communication, we show that the QCM measurement could be a powerful tool for the kinetic study of the reaction of the PHB depolymerase.

10.1021/bm0000844 CCC: $20.00 © 2001 American Chemical Society Published on Web 12/13/2000

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Figure 1. Schematic illustration of (A) the quartz crystal oscillator and (B) the QCM setup. Films are prepared on the gold electrode (0.189 cm2).

A schematic illustration of the QCM setup is shown in Figure 1. The QCM measurement system (SEIKO EG&G, QCA917) is modified by attaching the flow cell in which temperature is controlled by circulating water. The quartz crystal oscillator employed in this study is 9 MHz, AT-cut quartz, on both sides of which Au electrodes were deposited (area size, 0.189 cm2 × 2). According to the Sauerbrey equation,30 a frequency shift of 1 Hz corresponds to a mass change of 1 ng on the QCM electrode (0.189 cm2). In our system, the resonant frequency and admittance were monitored simultaneously to evaluate the influence of the friction change. Since no change of the admittance was observed during enzymatic reaction, friction change is negligible. On the one side of the oscillator, the films of poly[(S)2-hydroxypropionate] (P(2HP)), poly[(R)-3-hydroxybutyrate] (P(3HB)), poly[(R)-3-hydroxyoctanoate] (P(3HO)), polystyrene (PS), and polyethylene (PE) were initially prepared by conventional solvent-cast techniques from the chloroform solutions of P(2HP), P(3HB), P(3HO), and PS or from the p-xylene solution of PE, respectively. Then, the films were melted at a given temperature on a hot stage (Linkam LK600PM), and then the samples were kept at a given crystallization temperature for 3 days. After stabilization of the QCM in 0.01 M phosphate buffer solution (pH 7.4), enzymatic reaction was initiated by exchanging the buffer solution with the enzyme solution ([enzyme] ) 2.0 µg/mL). Figure 2A shows changes in the frequency (f) as a function of time during the enzymatic reaction of P(3HB) with the PHB depolymerase from Alcaligenes faecalis T1 at 37 °C. A negative shift in the frequency was observed just after the injection of the enzyme presumably because of enzyme adsorption to the surface of the P(3HB) film. Then, the frequency starts to increase within a few minutes indicating erosion of the film. The frequency kept on increasing until complete degradation of P(3HB) on QCM. Thus, the reaction of the PHB depolymerase can be

Figure 2. Time courses of frequency changes (∆F) and weight changes (∆w) obtained by the QCM, responding to the addition of (A) PHB depolymerase from A. faecalis T1 and (B) DFP-treated enzyme to P(3HB) film at 37 °C ([enzyme] ) 2.0 µg/mL). The arrows denote the starting points of the experiments.

followed continuously from enzyme adsorption to enzymatic degradation by the QCM technique. To evaluate individually the adsorption process of the enzyme, the active site in the catalytic domain of the PHB depolymerase was completely deactivated by treatment with diisopropyl fuluorophosphate (DFP).4 The frequency change measured during adsorption of the DFP-treated enzyme to P(3HB) film on QCM is shown in Figure 2B. Upon addition of the enzyme solution, a rapid frequency decrease was followed by the constant ∆f value of ca. -40 Hz. The transient response of the QCM to enzyme adsorption could not be observed because injection of the enzyme solution resulted in fluctuation of the signals for a few minutes presumably due to flux of the solution in the cell. The result indicates that the DFP-treated PHB depolymerase binds to the P(3HB) film within a few minutes. In previous papers,20,21 we investigated the adsorption equilibrium of GST fusion proteins with the substrate-binding domain of three bacterial PHB depolymerases and found that the adsorbing reaction of all GST-fusion proteins with powder and granule of P(3HB) reached to equilibrium within 5 min. These observations are consistent with the present result. When the sample is rinsed with the buffer solution after the frequency comes to a constant value, no frequency shifts were observed, indicating irreversible strong affinity of the depolymerase to the polyester films.

Communications

Figure 3. Time courses of frequency changes (∆F) and weight changes (∆w) observed for adsorption of PHB depolymerase from A. faecalis T1 (1.0, 2.0, and 4.0 µg/mL) to P(3HO) film at 37 °C. The arrow denotes the starting points of the experiments.

Figure 3 shows the frequency changes recorded on the injection of the PHB depolymerase solutions to P(3HO) film that is not hydrolyzed by the enzyme. The frequency decreased with time after the injection of the enzyme and came to a constant value within 20 min, indicating that only adsorption of the enzyme takes place on the surface of P(3HO) film and no erosion occurs. The adsorbed amount of the enzyme could be calculated from the frequency shift by calibration using the Sauerbrey equation.30 As shown in Figure 3, the amount of the adsorbed enzyme at equilibrium increased with enzyme concentration and attained a maximum value of ca. 40 ng under the condition of [enzyme] ) 2.0 µg/mL. At this maximum adsorbed amount, the apparent cross area per one molecule of the enzyme bound to the film surface is calculated to be ca. 40 nm2 by using a 48 kDa molecular mass of PHB depolymerase from A. faecalis T1,4,5 while the hydrodynamic radius of the depolymerase of 48 kDa molecular mass is evaluated to be approximately 3-4 nm on the assumption that the enzyme is globular.31 These facts suggest that the enzyme is packed on the surface of the film as monolayers at high enzyme concentrations. To elucidate the characteristics of the binding domain, we investigated the frequency changes during the course of enzyme adsorption on the four types of polymers that were not hydrolyzed by the PHB depolymerase from A. faecalis (Figure 4). Although no positive frequency shift by enzymatic hydrolysis was observed for all of four polymers, negative frequency shifts by enzyme adsorption were observed for P(2HP), P(3HO), and PS. The facts indicate that the catalytic domain and the binding domain operate independently. Although the minimum was observed for the P(2HP) curve in Figure 2, the origin of this behavior is unclear at present. To explain this phenomenon, not only kinetic study of the adsorption process but also investigation of the film surface would be need. Such detailed study is in progress in this laboratory. The rates of adsorption decreased in the following order: P(2HP) > P(3HO) > PS > PE. Interestingly, the binding domain has an affinity not only for various polyesters (PHAs) but also for polystyrene (PS), indicating that hydrophobic

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Figure 4. Time courses of frequency changes (∆F) and weight changes (∆w) observed for adsorption of PHB depolymerase from A. faecalis T1 (2.0 µg/mL) to various polymer films (P(2HP), P(3HO), PS, and PE) at 37 °C. The arrow denotes the starting points of the experiments.

interaction partially contributes to the affinity of the binding domain. Less affinity for PE than PS may suggest that the binding site of the enzyme recognizes the hydrophobic side chain of the polymer on the surface of the film. In the present study, a stronger affinity for PHAs than for polyolefines indicates that there is specific interaction between the ester bond of the substrate and the binding domain of the enzyme. Furthermore, this specific interaction seems to be hindered by a bulky alkyl group on the surface of the film as indicated by weaker affinity for P(3HO) as compared with P(3HB) and P(2HP). Thus, the PHB depolymerase might bind to a substrate by recognizing both the hydrophobic side chains and ester bonds on the surface of a polymer. References and Notes (1) Holmers, P. A. In DeVelopments in Crystalline Polymers, 2; Bassett, D. C., Ed.; Elsevier: London, 1988; p 1. (2) Doi, Y. Microbial Polyesters; VCH: New York, 1990. (3) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. (4) Tanio, T.; Fukui, T.; Shirakura, Y.; Saito, T.; Tomita, K.; Kaiho, T.; Masamune, S. Eur. J. Biochem. 1982, 124, 71. (5) Saito, T.; Suzuki, K.; Yamamoto, J.; Fukui, T.; Miwa, K.; Tomita, K.; Nakanishi, S.; Odani, S.; Suzuki, J.; Ishikawa K. J. Bacteriol. 1989, 171, 184. (6) Lusty, C. J.; Doudoroff, M. Proc. Natl. Acad. Sci. U.S.A. 1966, 56, 960. (7) Nakayama, K.; Saito, T.; Fukui, T.; Shirakura, Y.; Tomita, K. Biochim. Biophys. Acta 1985, 827, 63. (8) Jendrossek, D.; Mu¨ller, B.; Schlegel, H. G.; Eur. J. Biochem. 1993, 218, 701. (9) Jendrossek, D.; Frisse, A.; Behrends, A.; Andermann, M.; Kratzin, H. D.; Stanislawski, T.; Schlegel, H. G. J. Bacteriol. 1995, 177, 596. (10) Mukai, K.; Yamada, K.; Doi, Y. Polym. Degrad. Stab. 1994, 43, 319. (11) Ohura, T.; Kasuya, K.; Doi, Y. Appl. EnViron. Microbiol. 1999, 65, 189. (12) Mukai, K.; Yamada, K.; Doi, Y. Polym. Degrad. Stab. 1993, 41, 85. (13) Shinomiya, M.; Iwata, T.; Kasuya, K.; Doi, Y. FEMS Microbiol. Lett. 1997, 154, 89. (14) Kasuya, K.; Inoue, Y.; Tanaka, T.; Akehata, T.; Iwata, T.; Fukui, T.; Doi, Y. Appl. EnViron. Microbiol. 1997, 63, 4844. (15) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451. (16) Shiraki, M.; Iwata, A.; Saito, T. J. EnViron. Polym. Degrad. 1993, 2, 99. (17) Mukai, K.; Yamada, K.; Doi, Y. Int. J. Biol. Macromol. 1993, 15, 361.

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(18) Bachmann, B.; Seebach, D. Macromolecules 1999, 32, 1777. (19) Hiraishi, T.; Ohura, T.; Ito, S.; Kasuya, K.; Doi, Y. Biomacromolecules 2000, 1, 320. (20) Shinomiya, M.; Iwata, T.; Doi, Y. Int. J. Biol. Microbiol. 1998, 22, 129. (21) Kasuya, K.; Ohura, T.; Masuda, K.; Doi, Y. Int. J. Biol. Microbiol. 1999, 24, 329. (22) (a) Rodahl, M.; Ho¨o¨k, F.; Fredriksson, C.; Keller, C.; Krozer, A.; Brzezinski, P.; Voinova, M.; Kasemo, B. Faraday Discuss. 1997, 107, 229. (b) Ho¨o¨k, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12271. (c) Ho¨o¨k, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B. Langmuir 1998, 14, 729. (d) Fredriksson, C.; Kihlman, S.; Rodahl, M.; Kasemo, B. Langmuir 1998, 14, 248. (23) Laatikainen, M.; Lindstro¨m, M. J. Colloid Interface Sci. 1988, 135, 610. (24) Thompson, M.; Arthur, C. L.; Dahliwal, G. K. Anal. Chem. 1986, 58, 1206.

Communications (25) Rodahl, M.; Ho¨o¨k, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. ReV. Sci. Instrum. 1995, 66, 3924. (26) Muratsugu, M.; Ohta, F.; Miya, Y.; Hosokawa, T.; Kurosawa, S.; Kamo, N.; Ikeda, H. Anal. Chem. 1993, 65, 2933. (27) Janshoff, A.; Steinem, C.; Sieber, M.; Galla, H. J. Eur. Biophys. J. Biophys. Lett. 1996, 25, 105. (28) Caruso, F.; Furlong, D. N.; Kingshott P. J. Colloid Interface Sci. 1997, 186, 129. (29) Aberl, F.; Wolf, H.; Koesslinger, C.; Drost, S.; Woias, P.; Koch, S. Sen. Actuators, B 1994, 18-19, 271. (30) Saubrey, Z. Z. Phys. 1959, 155, 206. (31) Operator’s Manual of Dynamic Light Scattering Molecular Size Detectors (DynaPro-801 WIN and DynaPro-801TC); Protein Solutions Inc.

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