Degradation and Adsorption Characteristics of PHB Depolymerase As

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Degradation and Adsorption Characteristics of PHB Depolymerase As Revealed by Kinetics of Mutant Enzymes with Amino Acid Substitution in Substrate-Binding Domain Tomohiro Hiraishi,*,† Naoya Komiya,†,‡ Nobuhiko Matsumoto,§ Hideki Abe,§,| Masahiro Fujita,† and Mizuo Maeda*,†,‡ Bioengineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, Department of Advanced Materials Science, School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8568, Japan, Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama, Kanagawa 226-8501, Japan, and Chemical Analysis Team, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received August 24, 2009; Revised Manuscript Received November 4, 2009

Extracelluar Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZRpiT1) from Ralstonia pickettii T1 adsorbs to PHB surface via its substrate-binding domain (SBD) to enhance PHB degradation. Our previous study combining PCR random mutagenesis with the determination of PHB degradation levels of mutant enzymes suggested that Ser, Tyr, Val, Ala, and Leu residues in SBD are probably involved in the enzymatic adsorption to and degradation of PHB. In the present study, the effects of mutations at Leu441, Tyr443, and Ser445 on PHB degradation were investigated because these residues were predicted to form a β-sheet structure and orient in the same direction to interact possibly directly with the PHB surface. Purified L441H, Y443H, and S445C mutant enzymes were prepared, and their CD spectra and hydrolytic activities for water-soluble substrates were found to be identical to those of wild-type enzyme, indicating that these mutations have no influence on their structures and their ability to cleave the ester bond. In contrast, the PHB-degrading activity of these mutants differed from that of the wild type: L441H and Y443H enzymes had lower PHB-degrading activity than their wild-type counterpart, whereas S445C had higher activity. Kinetic analysis of PHB degradation by the mutants suggested that the hydrophobic residues at these positions are important for the enzyme adsorption to the PHB surface, and such substitutions as Y443H and S445C may more effectively disrupt the PHB surface to enhance the hydrolysis of PHB polymer chains than the wild-type enzyme. Surface plasmon resonance (SPR) analysis revealed that the three substitutions mentioned above altered the association phase rather than the dissociation phase in the enzyme adsorption to the polymer surface.

Introduction Poly[(R)-3-hydroxybutyrate] (PHB) is produced from and returned to renewable carbon sources. Accordingly, to sustain the environment, PHB has attracted academic and industrial interest as one of the alternative biopolyesters to petrochemical-based polymers.1 PHB is synthesized and accumulated as an intracellular carbon and energy storage compound in a wide variety of bacteria under natural environments.2 In the cells, PHB forms amorphous granules and is degraded by intracellular PHB depolymerases (i-PHB depolymerases) produced by the PHB-accumulating bacterium itself. In contrast, after PHB is extracted from the cells, PHB is converted to semicrystalline form and is degraded by extracelluar PHB depolymerases (e-PHB depolymerases) secreted from microorganisms in natural environments, such as soil, active sludge, fresh water, and seawater.3 In PHB recycling system, the discarded PHB materials are enzymatically degraded to oligomers or monomers by e-PHB depolymerases, and the resultant decomposed compounds are * Corresponding author. Tel: +81-48(467)9312. Fax: +81-48(462)4658. E-mail: [email protected] (T.H.); [email protected] (M.M.). † RIKEN Advanced Science Institute. ‡ The University of Tokyo. § Tokyo Institute of Technology. | RIKEN Advanced Science Institute.

utilized by prokaryotic and eukaryotic microorganisms.4,5 Finally, the compounds are converted into renewable resources, such as CO2 and biomass. Therefore, PHB decomposition process by e-PHB depolymerases is important as the first step of the PHB recycling process.6 A number of e-PHB depolymerases from microorganisms have been purified and characterized.3 Genetic analyses have shown that e-PHB depolymerases (multidomain e-PHB depolymerases) consist of a catalytic domain at the N-terminus, a substrate-binding domain (SBD) at the C-terminus, and a linker region connecting the two domains, although two exceptions (single-domain e-PHB depolymerases), PHB depolymerase from Penicillium funiculosum7 and PhaZ7 from Paucimonas lemoigne,8 have recently emerged. PHB degradation by multidomain e-PHB depolymerase is considered to proceed via a two-step reaction at the solid-liquid interface; that is, the enzyme approaches and adheres to the PHB surface via SBD, and the PHB polymer chain is hydrolyzed by the catalytic domain. Accordingly, the elucidation of the mechanisms of enzyme adsorption and enzymatic hydrolysis is expected to contribute to the development of new polymer materials with the desired stability and biodegradability as well as the development of improved e-PHB depolymerase that can be used to recycle effectively PHB materials.

10.1021/bm900967a  2010 American Chemical Society Published on Web 11/19/2009

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The structure-function relationship of e-PHB depolymerases has been studied extensively, and several mutants have been designed to analyze the function of SBD.9-11 Those studies have demonstrated that SBD is essential for the degradation of crystalline PHB material and not water-soluble substrates. In our recent studies using atomic force microscopy (AFM) with an SBD-immobilized tip, we found that SBD of e-PHB depolymerase from Ralstonia pickettii T1 (PhaZRpiT1) adsorbs specifically to the surface of such biopolyesters as PHB and poly(L-lactic acid) (PLLA) with an adhesive force of ∼100 pN.12,13 The structural aspects of an enzyme generally provide crucial information about the interaction between the enzyme and its ligand. Some researchers have reported the tertiary structures of polymer-degrading enzymes, such as glycoside hydrolases14 and single-domain e-PHB depolymerases,15,16 and proposed an interaction model between the enzymes and the polymer surfaces. However, because of the paucity of information about the 3D structures of multidomain e-PHB depolymerases, there is little insight into how amino acid residues in the SBD of e-PHB depolymerases are involved in the enzyme adsorption to the PHB surface and how the amino acid residues contribute to the adsorption. From a functional point of view, we have attempted to identify amino acid residues involved in PHB adsorption using PCR random mutagenesis that targets only SBD of PhaZRpiT1, in combination with an in vivo screening system.17 The results suggested that such amino acid residues as Ser, Val, Leu, Ala, and Tyr in SBD probably participate in the enzyme adsorption to the PHB surface. Nevertheless, because only little knowledge was obtained on the biochemistry and kinetics of the purified mutant enzymes, the roles of these amino acids in and their contributions to the enzymatic activity remain poorly understood, resulting in little information to develop e-PHB depolymerases. In the present study, we investigated the effects of L441H, Y443H, and S445C mutation in PhaZRpiT1 on the enzymes’ ability to adsorb to and degrade PHB because the residues at these positions form a β-sheet structure in its predicted secondary structure and orient in the same direction to probably interact with the polymer surface directly. To this end, we carried out a kinetic study of the degradation of PHB granules by these mutant enzymes and investigated how these amino acids contribute to the enzymatic adsorption and degradation. In addition, we examined in detail the kinetics of the adsorption of the mutant enzymes to the biopolyester surface using a surface plasmon resonance (SPR) apparatus.

Materials and Methods Materials, Reagents, and Bacterial Strains. Purified bacterial PHB of commercial grade was supplied by ICI, and its Mn and Mw/Mn were 189 000 and 2.6, respectively. PLLA was synthesized from L-lactide by ring-opening polymerization in the presence of diethylzinc/water catalyst, and its Mn and Mw/Mn were 12 000 and 1.9, respectively. All chemicals were of biochemical grade or the highest purity and were used without further purification. Restriction endonucleases and DNAmodifying enzymes were purchased from TAKARA, TOYOBO, and Roche. The enzymes were used in accordance with the supplier’s instructions. Escherichia coli JM109 (TAKARA) was used as host for the transformation with plasmids, and Rosetta-gami B (Novagen), which enhances the disulfide bond formation of target proteins, was used for the expression of PHB depolymerase mutants. Construction of Expression Plasmids. Three mutant genes (L441H, Y443H, and S445C) and the wild-type gene were amplified from pUCphaZRpiT1 plasmids17 having the corresponding mutant and wild-

Hiraishi et al. type genes by PCR with i-Cycler (BIO-RAD). Forward primer (5′CAACCACATATGGACGAGGGCGTAGGCGAG-3′) containing NdeI site (underlined) and reverse primer (5′-GAGGGATCCGATTTATTTATCTTCGAACAGC-3′) containing BamHI site (underlined) were used in the PCR reaction. The PCR products were digested with NdeI and BamHI and ligated into the same sites of expression plasmid pColdI (TAKARA) for the protein expression utilizing the promoter of coldshock gene. The resultant plasmids were named pColdIphaZRpiT1, and the insertion of phaZRpiT1 genes was confirmed by DNA sequencing. Enzyme Purification. Expression plasmids harboring wild-type and mutant phaZRpiT1 genes were transformed into E. coli Rosetta-gami B. Recombinant E. coli cells were grown in 1.0 L of Luria-Bertani medium containing 100 µg/mL ampicillin, 12.5 µg/mL tetracycline, and 20 µg/mL chloramphenicol at 37 °C to an O.D.600nm of 0.6, after which 0.1 mM IPTG (final concentration) was added to the culture medium. After the addition of IPTG, recombinant E. coli was cultured at 15 °C for 24 h. The cells were harvested by centrifugation at 3500g and 4 °C for 10 min. The collected cells were suspended in 5.0 mL of 10 mM phosphate buffer (pH 7.0) containing 0.3 M ammonium sulfate and disrupted by a French press four times at 14 000 psi. Crude cell extracts were centrifuged at 15 000g and 4 °C for 60 min, and the supernatant was filtered through 0.2 µm cellulose acetate filter. All purification procedures of the mutant and wild-type enzymes were carried out at 0-4 °C. The resultant filtrate was applied to TOYOPEARL Butyl-650S column (1.6 × 7.5 cm, TOSOH) that was pre-equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.3 M ammonium sulfate. The enzyme was eluted at a flow rate of 2.0 mL/min with a linear gradient of 0.3 to 0 M ammonium sulfate for three bed volumes, followed by a linear gradient of 0 to 40% ethanol for three bed volumes. The enzyme fractions were collected, dialyzed against 10 mM potassium phosphate buffer (pH 6.0), and applied to RESOURCE S column (0.64 × 3 cm, GE Healthcare). The enzyme was eluted at a flow rate of 1.0 mL/min with a linear gradient of 0 to 1 M NaCl for 50 bed volumes. The eluted enzyme fractions were dialyzed against 10 mM phosphate buffer (pH 7.0), concentrated, and stored at -30 or -80 °C. Enzyme Assays. The PHB-degrading activity of PhaZRpiT1 mutant enzymes was assayed as follows. A reaction mixture containing 0.4 g of PHB granules and 1.0 mM CaCl2 was prepared in 1.0 L of 50 mM Tris-HCl buffer (pH 7.5) and applied to ultrasonic treatment for the dispersion of the granules. The reaction was started by the addition of enzyme into 2.0 mL of the reaction mixture at 37 °C, and monitored at 600 nm with a MultiSpec-1500 spectrophotometer equipped with a temperature controller (Shimadzu). Degradation rates were determined by converting optical density into mass units (µg/min) using the linear correlation between optical density and PHB granule concentration (OD)1 is 150 µg/mL).18 To evaluate the effect of enzyme concentration on PHB degradation, we measured the reaction rates by setting the enzyme concentrations in the range of 0.5 to 4.0 µg/mL. Curve fitting was performed with GraFit software (Erithacus Software). Esterase activity was determined spectrophotometrically with paranitrophenylbutyrate (PNPB) as the water-soluble substrate. The reaction was started by the addition of enzyme solution (final concentration: 1.0 µg/mL) to 300 µL of the reaction mixture containing 0.5 mM PNPB in 10 mM phosphate buffer (pH 7.0) at 37 °C, and measurement was conducted at 405 nm with Wallac 1420 ARVOsx. We determined hydrolysis rates by converting optical density into mol units (µM/min) using the molar extinction coefficient (ε ) 14 000 M-1 cm-1).19 SPR Measurement. SPR measurement was performed with a Biacore T100 system (GE Healthcare). For the measurement, PLLA amorphous film was prepared on a gold-coated SPR sensor chip (SIA Kit Au, GE Healthcare) according to literature.13 All measurements were performed in 10 mM phosphate buffer (pH 7.0) at 37 °C under continuous flow at 20 µL/min. Sensorgrams were collected at enzyme concentrations of 0.25 to 1.0 µM. Data analysis was carried out with BIAevaluation 3.0 program.

Characteristics of PHB Depolymerase


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Figure 2. CD spectra of purified wild-type, L441H, Y443H, and S445C enzymes.

Figure 1. (A) Predicted secondary structure of PhaZRpiT1 SBD together with positions and frequencies of PCR-mediated single mutations,17 which possibly affect the phenotype of mutant enzymes in PHB degradation. Amino acid residues targeted here are indicated in bold letters. (B) Predicted hydropathy profiles of wild-type, L441H, Y443H, and S445C enzymes.

Analytical Procedures. Nucleotide sequences were determined with a CEQ2000XL sequencer (Beckman Coulter) using a Taq dye terminator cycle sequencing kit (Beckman Coulter). DNA and deduced amino acid sequences were analyzed with the sequence analysis program GENETYX (Software Development). Predictions of secondary and tertiary structures were performed with the programs SSpro8 and 3Dpro via SCRATCH20 web server, respectively. Hydrophobicity of the proteins was calculated with the parameters of Kyte and Doolittle.21 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) was performed according to the procedure of Laemmli22 with a molecular weight calibration kit (BIO-RAD). Protein was stained with Coomassie brilliant blue R250 (KANTO Chemical). Protein concentrations were determined by the method of Bradford23 with protein assay kit II (BIO-RAD), and bovine serum albumin was used as standard. Circular dichroism (CD) measurement was performed with a J-720 spectropolarimeter (JASCO). The far-UV spectra of the proteins were determined from 200 to 250 nm in 10 mM phosphate buffer (pH 7.0) at 20 °C with the following instrument settings: response, 1 s; sensitivity, 100 mdeg; and speed, 50 nm/min. The average of 30 scans was determined.

Results and Discussion Predicted Secondary Structure and Hydropathy Profiles of PhaZRpiT1 Mutants. In our previous study, the functional analysis of PhaZRpiT1 was accomplished with a combination of PCR random mutagenesis into its SBD and the determination of PHB degradation levels of recombinant E. coli.17 In the analysis of recombinants showing low PHB-degrading activity, Ser410, Tyr412, Val415, Tyr428, Ser432, Leu441, Tyr443, Ser445, Ala448, Tyr455, and Val457 were replaced with other residues at high frequency (Figure 1A). In addition, these amino acid residues tended to be replaced with residues having hydropathy indices opposite to theirs. This result suggested that the substitutions at these positions presumably inhibited the binding activity of PhaZRpiT1 to the PHB surface. Among these positions, Leu441, Tyr443, and Ser445 formed a β-sheet

structure to orient in the same direction on the basis of its predicted secondary structure, as shown in Figure 1A In general, such polymer-degrading enzymes as glycoside hydrolases14 and single-domain PHB depolymerases15,16 align their amino acid residues in a plane to interact with polymer surfaces. Among the mutations at these positions, the hydropathy indices of such mutations as L441H (replacement of Leu441 with His), Y443H (replacement of Tyr443 with His), and S445C (replacement of Ser445 with Cys) dramatically changed (Figure 1B). These findings implied that Leu441, Tyr443, and Ser445 residues in PhaZRpiT1 may directly interact with the PHB surface and motivated us to investigate the PHB-binding and -degrading properties of the three PhaZRpiT1 mutant enzymes with the substitutions mentioned above. Purification and Characterization of Mutant Enzymes. L441H, Y443H, and S445C enzymes as well as wild type were successfully purified from the soluble fraction of recombinant E. coli, as described in the Materials and Methods. The mutant enzymes showed the same binding properties during the protein purification procedure as their corresponding wild-type enzyme. To investigate whether their secondary structures were changed by the mutations, the CD spectra were measured. CD spectral analysis revealed that the spectra of wild-type, L441H, and Y443H enzymes were similar to those shown in Figure 2. In contrast, the CD spectrum of S445C was slightly different from that of wild type, but Cys may keep with the β-sheet structure because of its high potential for β-sheet forming.24 These findings suggested that their secondary structures remained significantly unchanged by the mutations. The esterase activities of L441H, Y443H, and S445C mutant enzymes were assayed to compare their abilities to cleave the ester bond with those of the wild-type enzyme. Figure 3 shows the PNPB hydrolysis rate of wild-type and mutant enzymes. PNPB hydrolysis rates of the mutant enzymes were comparable to that of the wild type. This result indicates that the function of the catalytic domain of PhaZRpiT1 is unaffected by these amino acid substitutions in SBD. Kinetics of PHB Degradation by Mutant Enzymes. To examine the effects of these amino acid substitutions in SBD on PHB degradation, we subjected the mutant enzymes to the assay for PHB degradation. Figure 4 shows typical timedependent changes in turbidity during the degradation of PHB granules by 0.5 µg/mL wild-type and mutant enzymes at 37 °C. In contrast with the results of PNPB hydrolysis, PHB degradation behaviors of the mutant enzymes were quite different from those of the wild type. L441H and Y443H enzymes had lower initial degradation rates (L441H: 19 ( 2.2

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Figure 3. PNPB hydrolysis rates of wild-type, L441H, Y443H, and S445C enzymes. The reaction was conducted at 37 °C in 10 mM phosphate buffer (pH 7.0) containing 1.0 µg/mL of the enzyme and 0.5 mM PNPB. Representative result of three independent experiments is shown.

Figure 5. Effects of PhaZRpiT1 concentration on the rate of PHB degradation by mutant and wild-type enzymes. (A) L441H (2), Y443H (9), and wild-type (b) enzymes. (B) S445C ([) and wild-type (b) enzymes. Enzymatic PHB degradation was performed at PhaZRpiT1 enzyme concentrations of 0.5 to 4.0 µg/mL. Representative result of three independent experiments is shown. Curves through the points represent the fit to each set of data with the equation, R ) ksK[E]/(1 + K[E]).2 Figure 4. Typical time-dependent changes in turbidity during PHB degradation at 37 °C by 0.5 µg/mL purified mutant and wild-type enzymes.

µg/min, Y443H: 18 ( 1.2 µg/min) than that of the wild type (24 ( 0.9 µg/min), whereas S445C enzyme had a higher initial degradation rate (32 ( 2.0 µg/min). Although it was previously reported that S445C enzyme may have reduced activity because of the low PHB-degrading level of recombinant E. coli cells,17 the PHB degradation rate of the purified S445C enzyme was unexpectedly higher than that of the wild type. These apparently conflicting results between the assay for PHB degradation and the previous in vivo assay with recombinant E. coli may be explained by the small amount of S445C enzyme secreted by recombinant E. coli cells. Taking into consideration the fact that the secondary structures and the PNPB-hydrolyzing activities of these mutant enzymes were retained, we suggest that the changes in PHB degradation by the mutant enzymes are due to changes in their adsorption to the PHB surface. The enzyme concentration dependence of the PHB degradation rates of mutant and wild-type enzymes was investigated at enzyme concentrations of 0.5 to 4.0 µg/mL. Figure 5 shows the effect of PHB depolymerase concentration on PHB granule degradation. When wild-type enzyme was used, the degradation rate increased with enzyme concentration, reaching a maximum at ∼1.0 µg/mL. At higher enzyme concentrations, the degradation rate decreased. This phenomenon could be explained in terms of the coverage change of the film surface by the adsorbed enzyme.25,26 The enzyme-concentration dependence of PHB degradation by L441H and Y443H mutant enzymes differed from that by the wild type (Figure 5A). The degradation rates

Table 1. Kinetic Parameters of PHB Degradation by PhaZRpiT1 Wild-Type and Mutant Enzymes enzyme

K (mL/µg)

ks (µg/min)

wild type L441H Y443H S445C

0.86 ( 0.01 0.39 ( 0.01 0.32 ( 0.01 0.95 ( 0.07

129 ( 3.6 138 ( 0.94 154 ( 0.94 152 ( 3.7

of these two mutant enzymes reached a constant value at enzyme concentrations of 2.0 to 4.0 µg/mL. The maximum degradation rate of L441H was 34 ( 0.7 µg/min, which is comparable to that of the wild-type enzyme (35 ( 0.5 µg/min), whereas Y443H enzyme showed a high degradation rate (38 ( 0.4 µg/min). In contrast with L441H and Y443H enzymes, S445C enzyme showed the highest degradation rate (39 ( 1.4 µg/min) at a low enzyme concentration of ∼1.0 µg/mL (Figure 5B). Kinetic studies of PHB degradation by the mutant enzymes were carried out to clarify the effects of mutation on enzyme binding and catalytic activities. Assuming that the adsorption of the enzyme to the polymer surface obeys a Langmuir isotherm, the degradation behavior could be well described by a heterogeneous kinetic model that consists of enzyme adsorption and subsequent hydrolysis of polymer chains.25,26 The equation can be written as R ) ksK[E]/(1 + K[E]),2 where R is the PHB degradation rate, ks is the surface hydrolysis rate constant, and K is the adsorption equilibrium constant of the enzyme. The present data points were analyzed with the abovementioned equation based on the heterogeneous model by using GraFit software. The kinetic parameters are listed in Table 1. The K values for L441H and Y443H enzymes are apparently lower than that for the wild-type enzyme, whereas that for S445C is slightly higher. The results suggest that the hydro-



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Table 2. Kinetic Parameters of the Adsorption Of PhaZRpiT1 Wild-Type and Mutant Enzymes to PLLA Surface

Figure 6. (A) SPR sensorgrams of the adsorption of wild-type (black), L441H (green), Y443H (blue), and S445C (orange) enzymes to the PLLA surface at the enzyme concentration of 0.5 µM. (B) Double logarithmic plots of ka versus kd for wild-type (black), L441H (green), Y443H (blue), and S445C (orange) enzymes.

phobic interactions at these positions of SBD positively contribute to the adsorption of the enzyme to the PHB surface. Interestingly, Y443H and S445C enzymes showed higher ks values than the other two enzymes, although the enzymatic degradation activities for soluble substrates, namely, the ability to cleave ester bonds, were identical among them. It is conceivable that the binding domain of polymer-degrading enzymes, such as PHB depolymerase27-29 and glycoside hydrolases,14 physically disrupts the polymer surface so that the active site of their catalytic domain can easily recognize the released polymer chains. Accordingly, the present finding suggests that such natural functions as the disruption of the PHB surface by SBD are possibly enhanced by Y443H and S445C substitutions in PhaZRpiT1. SPR Analysis of Adsorption of Mutant Enzymes to Biopolyester Surface. To clarify whether these amino acid substitutions (L441H, Y443H, and S445C) influence the association or dissociation processes in the interaction between SBD and PHB surface, kinetic analysis of enzyme adsorption to the biopolyester surface was performed by SPR measurement. PLLA was used as the substrate for SPR measurement because PLLA showed similar binding property for PhaZRpiT1 to PHB13 but was not degraded by PhaZRpiT1.28,29 Figure 6 shows typical SPR sensorgrams of the adsorption of PhaZRpiT1 enzymes to the PLLA surface at the enzyme concentration of 0.5 µM. When the enzyme solution was injected over the PLLA surface, a rapid signal response in the RU was observed because of the difference in refractive index between the buffer and the enzyme solution. This was followed by a slower increase that was indicative of enzyme adsorption to the polymer surface. After the continuous flow of the enzyme solution for 120 s, there was a rapid decrease due to refractive index switching caused by reinjection of the buffer. Following the rapid decrease, a slower decrease was observed as a result of the dissociation of the enzyme from the polymer surface. During the continuous flow of the enzyme solution, SPR sensorgrams of L441H and Y443H showed a slower increase

enzyme

ka (104 M-1 s1-)

kd (10-4 s1-)

KA (108 M-1)

wild type L441H Y443H S445C

11 ( 0.02 3.8 ( 0.02 4.1 ( 0.1 18 ( 0.05

6.5 ( 0.3 4.3 ( 0.5 4.7 ( 0.4 4.9 ( 0.4

1.7 0.88 0.88 3.6

than that of the wild type, whereas that of S445C showed the most rapid increase. At the end point of the enzyme injection, RU was in the order of S445C > wild type > Y443H > L441H, which was roughly in accordance with the order of the adsorption equilibrium constants of the enzymes on PHB degradation, as listed in Table 1. Curve fitting of the sensorgrams obtained here was performed with a 1:1 Langmuir binding model for both association and dissociation phases, but the sensorgrams were not well fitted by this model. In our previous work, curve fitting of the adsorption of PhaZRpiT1 SBD to the PLLA surface was improved by taking the effect of mass transfer into consideration.13 Therefore, the present sensorgrams were reanalyzed with the 1:1 Langmuir binding model with consideration of the mass transfer effect. The results are listed in Table 2. The kd values of the mutant enzymes were comparable to that of the wild type. The ka values of L441H and Y443H enzymes were approximately three times lower than that of the wild type, whereas that of S445C was approximately two times higher. To facilitate comparative analysis of the kinetic parameters, ka and kd, of the mutant enzymes, double logarithmic plots of ka versus kd were drawn using the values listed in Table 2 (Figure 6B). The results clearly show that the amino acid substitutions at positions 441, 443, and 445 mainly influence the association phase in the adsorption process of PhaZRpiT1 to the biopolyester surface; that is, the increase and decrease in hydrophobicity at these positions results in the improvement and deterioration of the ability of PhaZRpiT1 to approach to the polymer surface, respectively. Plausible Model of PHB Degradation by PhaZRpiT1 and Its Adsorption to PHB Surface. Figure 7A shows a plausible model of PHB degradation by PhaZRpiT1 wild-type enzyme. On the basis of the results of kinetic analysis of enzymatic PHB degradation and enzyme adsorption, first, the enzyme adsorbs to the PHB surface with kinetic parameters comparable to other biomolecular interactions, such as the ligand-receptor interaction.30 Previous AFM studies by Murase et al.27 and Kikkawa et al.28,29 have demonstrated that PhaZRpiT1 has the innate ability to disrupt the biopolyester surface via a noncatalytic process. Therefore, in the second stage, PhaZRpiT1 disrupts the PHB surface to solubilize the PHB chain in the liquid phase. It appears that this action allows the polymer chain to gain easy access to an active site in the catalytic domain of PhaZRpiT1. In the third stage, the released PHB chain is hydrolyzed to form monomers, oligomers, or both via the function of the catalytic domain. On the basis of the present findings, we propose a feasible process for PHB degradation by S445C mutant enzyme, as shown in Figure 7B. In the first stage, the association phase in the adsorption reaction of S445C enzyme is facilitated by the substitution of the hydrophilic residue with a hydrophobic one. The inset of Figure 7B shows a plausible model of the interaction between the residues at positions 441, 443, and 445 of S445C enzyme and the PHB surface, together with a predicted tertiary structure of SBD that depicts only the R carbon. The prediction of the tertiary structure of SBD was performed with 3Dpro program in which structural templates

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Figure 7. Proposed mechanism of PHB degradation by PhaZRpiT1 (A) wild-type and (B) S445C enzymes on the basis of the present kinetic studies. Inset shows a plausible model of the interaction between amino acid residues at positions 441, 443, and 445 in SBD of S445C enzyme and the PHB polymer chain. A predicted tertiary structure of SBD depicts only the R carbon, and the R carbons at positions 441, 443, and 445 in SBD of S445C are indicated in green, blue, and orange spheres, respectively.

are not used. The predicted 3D structure suggested that the above-mentioned residues are located on a planar surface of SBD and seem to interact directly with the PHB surface. Taking the present kinetic results and the structural findings into consideration, we surmised that the hydrophobic residues at positions 441, 443, and 445 of PhaZRpiT1 S445C enzyme play a crucial role in the induction of the interaction between SBD and the PHB surface. This appears to be consistent with the previous structural viewpoints on single-domain PHB depolymerases of Hisano et al.15 and Papageorgiou et al.16 They suggested that hydrophobic and aromatic residues exposed to the solvent presumably interact with the PHB polymer surface via hydrophobic interaction. In the second stage of PHB degradation, PHB surface disruption is enhanced by the replacement of Ser445 with Cys, although the reason for this enhancement is still unknown. Finally, hydrolysis of the PHB chain by S445C enzyme proceeds at a rate comparable to that by the wild type. Therefore, the present findings permit us to speculate that the replacement of hydrophilic amino acid residues with hydrophobic ones such as S445C might promote not only the adsorption of PhaZRpiT1 to the PHB surface but also the disruption of the PHB surface to release its polymer chains to the solvent.

Conclusions Effects of amino acid substitutions at the positions of 441, 443, and 445, at which the residues possibly form β-sheet structure to interact with the polymer surface directly, in

PhaZRpiT1 on the enzymes’ ability to adsorb to and degrade PHB were kinetically studied. Among the previously obtained mutations at these positions, L441H, Y443H, and S445C mutations were investigated because their hydropathy indices dramatically changed compared with those of wild type. Purified L441H, Y443H, and S445C mutant enzymes had identical CD spectra and hydrolytic activities for water-soluble substrates with those of wild type, indicating that these mutations have no influence on their structures and their abilities to cleave the ester bond. In contrast, their PHB degradation abilities were affected by the mutations; that is, L441H and Y443H enzymes had lower PHB-degrading activity than their wild-type counterpart, whereas S445C had higher activity. Kinetic analysis of PHB degradation by the mutant enzymes suggested that the hydrophobic interactions at these positions of SBD positively contribute to the adsorption of the enzyme to the PHB surface. Interestingly, such mutations as Y443H and S445C possibly enhanced the disruption of PHB surface. SPR analysis of the adsorption of PhaZRpiT1 mutant enzymes to PLLA surface indicated that the amino acid replacements at the positions of 441, 443, and 445 influence mainly the association phase rather than the dissociation phase in the adsorption process of PhaZRpiT1 to the biopolyester surface. Acknowledgment. This research was supported by a Grantin-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 18750138) (to T.H.), Incentive Research Grant (to T.H.) from RIKEN Institute, and grants for Ecomolecular Science Research and Clean Chemistry Research from RIKEN Institute.


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BM900967A

Degradation and Adsorption Characteristics of PHB Depolymerase As

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