Characterization of an Extracellular Medium-Chain-Length Poly (3

for production of extracellular MCL-PHA depolymerases. Bong Su Seo , Do Young Kim , Yu-Yang Ni , Kwang-Hee Son , Ho-Yong Park , Young Ha Rhee...
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Biomacromolecules 2002, 3, 291-296

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Characterization of an Extracellular Medium-Chain-Length Poly(3-hydroxyalkanoate) Depolymerase from Pseudomonas alcaligenes LB19 Do Young Kim, Jin Sik Nam, and Young Ha Rhee* Department of Microbiology, Chungnam National University, Daejeon 305-764, Korea Received July 4, 2001; Revised Manuscript Received November 11, 2001

An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa. The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 °C. The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol. It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100. The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide). Biodegradation rates of the aromatic MCLPHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents. It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms. The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units. The results demonstrated in this study suggest that the MCL-PHA depolymerase from P. alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA. Introduction Polyhydroxyalkanoates, PHAs, are environmentally friendly polymers that are biosynthesized and accumulated intracellularly by numerous microorganisms.1 The ability to decompose extracellular PHA and to utilize the degradation products as sources of carbon and energy depends on the secretion of specific extracellular PHA depolymerase, which hydrolyzes the PHA to water-soluble products.2 Extracellular PHA depolymerases are divided into two groups according to their substrate specificity.3 One group is short-chain-length (SCL) PHA depolymerases that degrade poly(3-hydroxybutyrate) and/or its copolyesters with 3-hydroxyvalerate. The other group is composed of medium-chain-length (MCL) PHA depolymerases that decompose polymers consisting of 3-hydroxyalkanoates with six or more carbon atoms. A number of aerobic and anaerobic PHA-degrading microorganisms including bacteria and filamentous fungi have been isolated from various ecosystems, and as a result several extracellular PHA depolymerases have been purified and characterized. However, most of the PHA depolymerases identified were specific for only SCL-PHAs.3 In contrast, reports on microorganisms capable of degrading MCL-PHAs in the environment are relatively rare.4-6 Only three MCLPHA depolymerases from Pseudomonas fluorescens GK13,7 Pseudomonas sp. RY-1,8 and Xanthomonas sp. JS029 have * To whom correspondence may be addressed. Phone: (8242) 821-6413. Fax: (8242) 822-7367. E-mail: [email protected].

been isolated and characterized to date. However, the poly(3-hydroxyoctanoate) depolymerase from P. fluorescens GK13 is the only MCL-PHA depolymerase that has been characterized to the molecular level.5,7,10 Thus, the enzymatic properties of MCL-PHA depolymerases are not well documented. In this research, we describe the isolation of a MCL-PHA degrading bacterium, P. alcaligenes LB19, and the purification and some properties of the MCL-PHA deploymerase produced by the isolate. Exceptional properties of this enzyme are emphasized and compared with those of MCLPHA depolymerase from other microbial origins. Experimental Section Preparation of Polyesters and Their Latex Suspensions. Aliphatic and aromatic MCL-PHAs were produced by culturing P. oleoVorans ATCC 29347 and P. putida KCTC 2407 in a growth medium containing the carbon substrates with corresponding chemical structure as described in Table 1. Synthesized PHAs were isolated and purified from lyophilized cells by extraction with hot chloroform using a Soxhlet apparatus as described previously.11 Poly(3-hydroxybutyrate) (PHB) was produced from Ralstonia eutropha and isolated by simple solubilization of the cells harvested from culture broth by autoclaving a lytic solution (pH 9.0) containing 2 g of cells, 1 g of EDTA, and 2 g of sodium dodecyl sulfate in 100 mL of distilled water; polymer

10.1021/bm010113q CCC: $22.00 © 2002 American Chemical Society Published on Web 02/07/2002

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Table 1. Enzymatic Degradabilities of Various Aliphatic and Aromatic PHAs by the MCL-PHA Depolymerase from P. alcaligenes LB19 microorganisms

R. eutropha P. putida P. putida P. putida P. putida P. oleovorans P. oleovorans P. oleovorans P. putida P. putida P. putida

carbon substratesa (mol %) butyrate (100) heptanoate (100) octanoate (100) nonanoate (100) decanoate (100) 10-UND(d) (100) 10-UND(d) (50) + 10-UND(t) (50) 5-PV (100) 8-p-MPO (100) 5-PV (50) + 8-p-MPO (50) 6-PHx (50) + 8-p-MPO (50)

synthesized PHAs

depolymerase activityd

P(68.8% 3HHp-co-19.1% 3HN-co-12.1% 3HUD)b P(4.4% 3HHx-co-86.0% 3HO-co-9.6% 3HD)b P(25.3% 3HHp-co-74.7% 3HN)b P(3.3% 3HHx-co-62.5% 3HO-co-34.2% 3HD)b P(7.5% 3HHp(d)-co-61.7% 3HN(d)-co-30.8% 3HUD(d))b P(8.6% 3HHp(d)-co-29.6% 3HN(d)-co-38.8% 3HN(t)-co8.6% 3HUD(d)-co-12.6% 3HUD(t))b P(3H(5PV))b P(28.5% 3H(4pMPB)-co-71.5% 3H(6pMPHx))c P(65.0% 3H(5PV)-co-6.7% 3H(4pMPB)-co-28.3% 3H(6pMPHx))c P(3.9% 3H(4pMPB)-co-36.1% 3H(6pMPHx)-co-21.9% 3H(4PB)-co38.1% 3H(6PHx))c

+++ +++ +++ ++++ +++ +++

P(3HB)b

+ + + +

a Key: 10-UND(d), 10-undecenoate; 10-UND(t), 10-undecynoate; 5-PV, 5-phenylvalerate; 8-p-MPO, 8-p-methylphenoxyoctanoate; 6-PHx, 6-phenoxyhexanoate. b GC area %. c Mole percent determined by 1H NMR spectroscopy. 3HB, 3-hydroxybutyrate; 3HHx, 3-hydroxyhexanoate; 3HHp, 3-hydroxyheptanoate; 3HO, 3-hydroxyoctanoate; 3HN, 3-hydroxynonanoate; 3HD, 3-hydroxydecanoate; 3HUD, 3-hydroxyundecanoate; 3HHp(d), 3-hydroxy6-heptenoate; 3HN(d), 3-hydroxy-8-nonenoate; 3HUD(d), 3-hydroxy-10-undecenoate; 3HN(t), 3-hydroxy-8-nonynoate; 3HUD(t), 3-hydroxy-10undecynoate; 3H(5PV), 3-hydroxy-5-phenylvalerate; 3H(4PB), 3-hydroxy-4-phenoxybutyrate; 3H(6PHx), 3-hydroxy-6-phenoxyhexanoate; 3H(4pMPB), 3-hydroxy-4-p-methylphenoxybutyrate); 3H(6pMPHx), 3-hydroxy-6-p-methylphenoxyhexanoate. d Depolymerase activity was determined by measuring the reaction time taken for each polymer in the standard assay mixture (initial O.D., 1.0 ( 0.02) to be completely degraded: ++++ (within 20 min); +++ (within 30 min); + (within 20 h).

granules were subsequently collected by centrifugation. To remove the remaining contaminants the polymer granules were washed twice with methanol, and rewashed with cold water. The fine polymer particles were then recovered by centrifugation. Poly(L-lactide) (PLA; number average molecular weight, 130 000) and polycaprolactone (PCL; number average molecular weight, 80 000) were purchased from the Sigma Chemical Co. and the Aldrich Chemical Co., respectively. Latex suspensions of MCL-PHAs and PCL were prepared according to the method described by Ramsay et al.12 with minor modifications. Briefly, four volumes of cold water were carefully poured into one volume of polymer solution in acetone with stirring, and the acetone was then evaporated. To prepare the PLA latex, 4 volumes of cold water were initially poured into 1 volume of PLAchloroform solution, and the colloidal PLA was then obtained by ultrasonic treatment followed by evaporation of the chloroform. A suspension of PHB was made by ultrasonically dispersing PHB granules in distilled water. The number average molecular weights and polydispersity indices of all aliphatic MCL-PHAs and the PHA containing phenyl groups were approximately 50 000 and 2.5 ( 0.2, respectively, regardless of the polymer types, as determined by gel permeation chromatography. The number average molecular weight of the PHA produced by P. putida from 8-pmethylphenoxyoctanoate was 25 000 and that of the PHAs synthesized from a equimolar mixture of 8-p-methylphenoxyoctanoate and 5-phenylvalerate or 6-phenoxyhexanoate was approximately 33 000. Isolation and Identification of a MCL-PHA-Degrading Microorganism. A MCL-PHA-degrading bacterial strain was isolated from a soil sample by enrichment using a mineral salt agar medium13 containing 0.1% (wt/v) polyhydroxynonanoate (PHN). The nutritional characteristics of the isolate were determined according to ref 14, and sequence analysis of the 16S rRNA gene of the isolate was performed as previously described.15 The 16S rDNA sequences were aligned with those from strains of the genus Pseudomonas,

on the basis of similarities in the primary and secondary RNA structures using the PHYDIT program.16 Production of MCL-PHA Depolymerase. To obtain the enzyme, the strain LB19 was first grown by shaking on a rotary shaker at 200 rpm for 18 h at 30 °C using six 2-L Erlenmeyer flasks, which each contained 500 mL of nutrient broth. Cells were harvested by centrifugation and transferred to a fermentor containing a mineral salt medium supplemented with 600 mL of PHN latex (1%, w/v). Each liter of mineral salt medium contained 0.66 g of (NH4)2SO4, 7.30 g of Na2HPO4‚12H2O, 2.30 g of KH2PO4, 0.30 g of NaHCO3, 0.25 g of MgSO4‚7H2O, 0.01 g of CaCl2‚2H2O, and 1 mL of trace element solution. The trace element solution contained 0.58 g of ZnSO4‚7H2O, 3.96 g of MnCl2‚4H2O, 0.60 g of H3BO4, 5.56 g of FeSO4‚7H2O, 5.62 g of CoSO4‚ 7H2O, 0.34 g of CuCl2‚2H2O, 0.04 g of NiCl2‚6H2O, and 0.06 g of Na2MoO4‚2H2O per liter of 0.5 N HCl. The fermentation experiments were conducted in a 5-L jar fermentor (Korea fermentor Co. Ltd) with a working volume of 3 L for 24 h. The temperature and pH were automatically controlled at optimal values of 30 °C and 7.0, respectively. The airflow rate was set at 0.1 vvm, and the agitation speed was controlled in the range 80-200 rpm to reduce the aggregation of polymer during fermentation. Purification of MCL-PHA Depolymerase. The culture supernatant was applied onto an Octyl-Sepharose CL-4B column (90 mL bed volume; 24 mm diameter) preequilibrated with 50 mM glycine-NaOH buffer, pH 9.0, and the enzyme was eluted with a gradient of 0-50% ethanol. The fractions with high MCL-PHA depolymerase activity were collected, concentrated 10- to 15-fold by ultrafiltration using a PM10, and then applied onto a Sephadex G-150 column (320 mL bed volume; 28 mm diameter) equilibrated with the same buffer. The fractions containing MCL-PHA depolymerase were pooled and concentrated using a PM10 membrane. Enzyme Assays. MCL-PHA depolymerase activity was routinely assayed by measuring the turbidity decrease at 650

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nm of a PHN latex. The reaction mixture containing 150 µL of PHN latex, 50 µL (30 mU) of the enzyme, and 50 mM of glycine-NaOH buffer (pH 9.0) in a total volume of 2 mL was incubated at 37 °C for 15 min. The initial optical density of the reaction mixture was in the range of 1.0 ( 0.02. One unit of MCL-PHA depolymerase activity was defined as decrease of the value of A650 by 1 unit/min. Esterase activity was assayed in 2 mL of 50 mM glycineNaOH buffer, pH 8.0, using various p-nitrophenylalkanoates (PNP-alkanoates). The reaction mixtures contained 30 µL of a 10 mM solution of the respective PNP-alkanoates in ethanol and 25 µL (15 mU) of the enzyme solution. Enzyme Analysis. A quaternary structure of the native enzyme was determined by gel permeation chromatography using Sephacryl S-200. Cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa), albumin (66.0 kDa), and alcohol dehydrogenase (150.0 kDa) were used as molecular mass standards. The relative molecular mass (Mr) of the denatured MCL-PHA depolymerase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an 11% gel according to the method described by Laemmli.17 Serum albumin (66.0 kDa), fumarase (48.5 kDa), carbonic anhydrase (29.0 kDa), β-lactoglobulin (18.4 kDa), and R-lactalbumin (14.2 kDa) were used as molecular mass standards. Isoelectric focusing was performed using PhastGel slabs (pH 3 to 9). Glucose oxidase (pI, 4.2), trypsin inhibitor (pI, 4.6), β-lactoglobulin (pI, 5.1), carbonic anhydrase II (bovine; pI, 5.9), and carbonic anhydrase I (human; pI, 6.6) were used as pI standards. Protein concentrations were measured by Bradford’s method18 using bovine serum albumin as the standard. Determination of Hydrolysis Products. To identify the hydrolysis products, 7 mL of a reaction mixture containing approximately 5 mg of colloidal PHN, the purified MCLPHA depolymerase (100 mU), and glycine-NaOH buffer (pH 9.0) were initially incubated at 37 °C for 12 h. Thereafter, 50 µL of a 5 M HCl solution was added to the reaction mixture to stop the reaction, and at this point the reaction mixture’s pH was 2.1. The hydrolysis products were extracted with ethyl acetate within 3 min according to the method described by Kita et al.19 Ethyl acetate was removed by evaporation at 70 °C and then the hydrolysis products were dissolved in methylene chloride and identified by GC and GC/MS as described elsewhere.20 Nucleotide Sequence Accession Number. The 16S rDNA sequence of the strain LB19 has been deposited in GenBank under accession no. AF390747. Results Identification and Characteristics of the Isolate. The isolate was a Gram-negative rod, motile, and non-sporeforming bacterium. It utilized organic compounds containing glucose, citrate, benzoate, lactate, succinate, fumarate, the alkanoic acids of acetate to nonanoate, and several amino acids as carbon sources. However, glycine, L-phenylalanine, fructose, mannitol, sorbitol, gluconate, levulinate, β-hydroxybutyrate, and starch did not support the growth of LB19. These results showed that the carbon requirements of strain LB19 are almost identical to that of P. alcaligenes, as

Table 2. Purification of the MCL-PHA Depolymerase from P. alcaligenes LB19

purification step supernatant Octyl-Sepharose CL-4B Sephadex G-150

total total specific protein activity activity (mg) (U) (U/mg) 30.0 3.7 0.8

224.2 201.4 142.4

7.4 54.7 178.0

yield (%)

purification fold

100.0 89.6 63.5

1.0 7.4 24.0

described in ref 14, with the exception of glucose utilization. Phylogenetic analysis of the nucleotide sequences of the 16S rDNA showed that the strain LB19 was closely linked with P. alcaligenes LMG 1224T, with a highest sequence similarity of 99.2%. From these results, the isolated strain LB19 was identified as P. alcaligenes and deposited in the Korean Collection for Type Cultures under code no. P. alcaligenes KCTC 12029. Purification of MCL-PHA Depolymerase. To improve the purification efficiency of the MCL-PHA depolymerase from P. alcaligenes LB19, we applied the whole culture supernatant directly onto an Octyl-Sepharose CL-4B column. Pretests showed that this procedure, when used as the first step of the purification, was more efficient with respect to the yield of the purified enzyme than the traditional method, which involves proteins being either concentrated by ammonium sulfate precipitation or ultrafiltered prior to chromatography. MCL-PHA depolymerase was strongly bound to hydrophobic materials in the column and was eluted by a gradient of approximately 30-35% ethanol. For further purification the proteins obtained from hydrophobic interaction chromatography were repurified by gel permeation chromatography using Sephadex G-150. A quantitative evaluation of the results obtained from the consecutive purification steps is listed in Table 2. The enzyme was purified 24-fold with an overall yield of 63.5%. Molecular Mass and pI. The molecular mass of the native enzyme was estimated to be 28.0 kDa by Sephacryl S-200 column chromatography (Figure 1). Interestingly, despite electrophoretic homogeneity in native polyacrylamide gel, the purified enzyme, even in SDS-polyacrylamide gel containing 5 M urea, was separated into two polypeptide bands, one major band with a Mr of 26.5 kDa and a second minor band with a Mr of 27.5 kDa (Figure 2a). The Mr of the larger polypeptide was nearly identical with the molecular mass of the native enzyme. The molecular mass (27 668 Da) of the enzyme, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, was also similar to that of the larger polypeptide. Recently, a similar phenomenon was also observed in the PHB depolymerase from Marinobacter sp.21 In that case, formation of a polypeptide chain with a truncated C-terminus was observed during hydrophobic interaction column chromatography. The pI value of the MCL-PHA depolymerase from P. alcaligenes LB19 was determined to be 5.7 by isoelectric focusing (Figure 2b). Effects of pH and Temperature on Enzyme Activity. The MCL-PHA depolymerase showed over 60% of its maximum activity in the pH range 8.0-9.5 and was drastically inactivated at pH values above 10.5. The highest activity of the enzyme was obtained in 50 mM glycineNaOH buffer, pH 9.0. The enzyme was fairly stable at pH

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Kim et al. Table 3. Effects of Various Inhibitors on MCL-PHA Depolymerase Activitya

Figure 1. Gel filtration chromatography of the purified MCL-PHA depolymerase from P. alcaligenes LB19 using Sephacryl S-200. The enzyme was indicated by the arrow. Cytochrome c (12.4 kDa), carbonic anhydrase (29.0 kDa), albumin (66.0 kDa), and alcohol dehydrogenase (150.0 kDa) were used as molecular mass standards.

Figure 2. SDS-PAGE (a) and isoelectric focusing (b) of the purified MCL-PHA depolymerase from P. alcaligenes LB19. Proteins were separated on a SDS-11% polyacrylamide gel (a) and a PhastGel slab (pH 3-9) (b), and silver stained: lane 1, purified enzyme (0.6 µg); lane 2, molecular mass and pI standards.

values between 6.0 and 10.5 and retained more than 95% of its original activity even after 1 h of preincubation at these pH values. The optimum temperature of the enzyme was 45 °C, and it retained approximately 88% of its maximum activity even at 50 °C. The MCL-PHA depolymerase was relatively stable for 1 h at the temperature below 45 °C but was inactivated to less than 10% of its original activity when it was exposed at 55 °C for the same period of time. Effects of Protein Inhibitors on Enzyme Activity. The inhibitory effects of various types of protein inhibitors on MCL-PHA depolymerase activity are listed in Table 3. Dithiothreitol, which reduces disulfide bonds, and carbodiimide, a carboxyl-group-directed inhibitor, had no effect on the enzyme activity after 1 h of preincubation at 37 °C. On the other hand, it was significantly inhibited by 2 mM of p-hydroxymercuribenzoic acid. The activity of the MCLPHA depolymerase was also markedly decreased by other sulfhydryl reagents, such as sodium azide, N-ethylmaleimide, and iodoacetamide, at a concentration of 10 mM. Inhibition

inhibitor

concn (mM)

residual activity (%)

none DTT sodium azide NEM IAA PHMB NBS carbodiimide acetic anhydride PMSF EDTA

10 10 10 10 2 5 10 3 2 1

100 100 13 6 35 15 5 100 17 16 8

a The reaction mixture (1.97 mL) containing enzyme 25 µL (15 mU) of the enzyme solution, inhibitor, and 50 mM glycine-NaOH buffer (pH 8.0) was initially preincubated for 1 h at 37 °C, and then the enzymatic reaction was started by adding 30 µL of a 10 mM solution of PNP-octanoate. DTT, dithiothreitol; NEM, N-ethylmaleimide; IAA, iodoacetamide; PHMB, phydroxymercuribenzoic acid; NBS, N-bromosuccinimide; PMSF, phenylmethylsulfonyl fluoride.

of the enzyme activity by N-bromosuccinimide and acetic anhydride indicated that tryptophan and lysine residues play an important role in the catalytic domain of the enzyme. Of the protein inhibitors evaluated, EDTA proved toxic enough to inhibit more than 90% of the original activity of the MCLPHA depolymerase even at a concentration of only 1 mM. The inactivation of the enzyme by phenylmethylsulfonyl fluoride (PMSF) strongly suggested the presence of serine residues in its active site. Moreover, the purified enzyme was completely inhibited by nonionic detergents such as 0.1% Tween 80 and 0.05% Triton X-100, indicating that a hydrophobic region may be located near or at the active site. When the depolymerase was treated with 1 mM of divalent cations such as Mn2+, Zn2+, and Ag2+, its hydrolysis activity of PNP-octanoate at pH 7.5 was significantly inhibited, while it was practically insensitive to Ca2+ and Mg2+. It was also partially inhibited by metal ions such as Co2+, Cu2+, Hg2+, Ni2+, and Fe2+at a concentration of 1 mM. Substrate Specificities and Hydrolysis Products. Biodegradabilities of various polymers by the purified MCLPHA depolymerase from P. alcaligenes LB19 were evaluated. Polymers tested and their compositions are listed in Table 1. The enzyme could practically hydrolyze all aliphatic and aromatic MCL-PHAs but not PHB, PLA, and PCL. However, in this study, biodegradabilities of polymers by this enzyme could not be quantitatively compared by measuring turbidity decrease in the reaction mixtures due to the different light scattering of each polymer. Thus, the biodegradability of polymers was evaluated by an indirect observation that measures the reaction time being required for decreasing of 1 absorbance in the reaction mixture. Hydrolysis rates of the aliphatic MCL-PHAs consisting of saturated and/or unsaturated repeating units by the enzyme from P. alcaligenes LB19 were always significantly higher (approximately 40-60 times) than those of the aromatic PHAs bearing phenyl, phenoxy, and p-methylphenoxy groups. Especially, in the case of the MCL-PHAs synthesized from heptanoic to decanoic acids, it was estimated that biodegradability of polyhydroxydecanoate (PHD) was the highest. The experimental results on biodegradation of these MCL-PHAs determined by a clear zone tube method also

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monomers were identified as the major products after the enzymatic hydrolysis of PHN for 12 h, dimers were markedly decreased, and trimers absent. Discussion

Figure 3. Biodegradation of saturated aliphatic MCL-PHAs in clear zone tubes by P. alcaligenes LB19 grown at 30 °C for 7 days. PHH homopolymer was produced by Pseudomonas sp. HJ-215 from heptanoate, and other MCL-PHA copolymers were obtained from P. putida grown with heptanoate to decanoate. Table 4. Substrate Specificities of the MCL-PHA Depolymerase from P. alcaligenes LB19 substrate

esterase activity ((µmol of p-nitrophenol/min)/mg of protein)

PNP-acetate PNP-butyrate PNP-hexanoate PNP-octanoate PNP-decanoate PNP-dodecanoate PNP-hexadecanoate

0.1 0.3 2.8 6.1 6.2 6.8 3.1

indicated that the degradability of PHD was better than that of other polymers (Figure 3). It should be noted that the remarkable difference of biodegradation rates of the aromatic PHAs by the enzyme was not observed, despite the compositions of PHAs and the types of functional groups in the side chains were significantly different. The apparent Km and Vmax values of the MCL-PHA depolymerase from P. alcaligenes LB19 for PHO and PHN were 0.32 mg/mL and 2000 U/mg and 0.41 mg/mL and 330 U/mg, respectively. The catalytic efficiency (Vmax/Km) for PHO was higher approximately 7.8-fold that for PHN. Of the PNP-alkanoates tested, PNP-dodecanoate was hydrolyzed most efficiently by the enzyme from P. alcaligenes LB19 (Table 4). On the other hand, the esterase activities of the enzyme for PNP-acetate and PNP-butyrate were significantly lower. It was also able to hydrolyze PNP-palmitate, which is a suitable substrate for lipase. GC chromatograms showed that the composition and relative amounts of hydrolysis products of PHN were significantly dependent upon the reaction time. The heterodimer of 3HHp and 3HN was detected as the main hydrolysis product during the early enzymatic reaction period (within about 1 h) with smaller amounts of the 3HHp and 3HN monomers and the corresponding trimers. However, oligomers above tetramer were undetected. In contrast,

Over the past decade, much intensive effort has been spent upon the preparation and application of MCL-PHAs with improved properties.22,23 However, relatively few reports have been published on microorganisms and their MCL-PHA depolymerases, with respect to the biodegradation of these polymers.4,6,7-9 We describe herein the distinct biochemical properties of the purified MCL-PHA depolymerase from P. alcaligenes LB19, which are quite distinct from those of other MCL-PHA depolymerases. The quaternary structure and molecular mass of the MCLPHA depolymerase (monomer, 27.6 kDa) from P. alcaligenes LB19 are significantly different from those of the PHO depolymerase (dimer, 48 kDa) from P. fluorescens GK13,7 the MCL-PHA depolymerase (tetramer, 115 kDa) from Pseudomonas sp. RY-1,8 and the poly(3-hydroxy-5-phenylvalerate) (PHPV) depolymerase (monomer, 41.7 kDa) from Xanthomonas sp. JS02.9 In contrast to the general property of PHA depolymerases, the MCL-PHA depolymerase from P. alcaligenes LB19 showed the pI value in the acidic pH range (Figure 2b). Similar observations were also made for the MCL-PHA depolymerase from Pseudomonas sp. RY-18 and the PHB depolymerase from Alcaligenes faecalis AE122.19 However, it has been reported that the PHO depolymerase from P. fluorescens GK137 has an alkaline pI value as do the majority of SCL-PHA depolymerases. The optimum pH (9.0) of the MCL-PHA depolymerase from P. alcaligenes LB19 is relatively similar to that (8.5) of the other MCL-PHA depolymerases reported. The optimum temperature (45 °C) of this enzyme is also identical with that of the PHO depolymerase from P. fluorescens GK13,7 while it is much lower than that of PHPV depolymerase (60 °C) from Xanthomonas sp. JS029 and higher than that of the MCL-PHA depolymerase (35 °C) from Pseudomonas sp. RY-1.8 The MCL-PHA depolymerase from P. alcaligenes LB19 is believed to be an esterase belonging to the serine hydrolase family, like other PHA depolymerases, because it is markedly inhibited by PMSF (Table 3). However, neither the PHO depolymerase from P. fluorescens GK137 nor the MCL-PHA depolymerase from Pseudomonas sp. RY-18 is inactivated by PMSF. The MCL-PHA depolymerase from P. alcaligenes LB19 is a metalloprotein that requires metal ions as a cofactor for catalytic activity, because it was drastically inactivated by EDTA even at a concentration of 1 mM (Table 3). The MCL-PHA depolymerase from Pseudomonas sp. RY-1 was also significantly inhibited by EDTA,8 while the PHO depolymerase from P. fluorescens GK13 was relatively insensitive to the same inhibitor.7 It is of interest that all MCL-PHA depolymerases produced from the three Pseudomonas species were not inhibited by DTT unlike most SCLPHA depolymerases.24-27 These results suggest that disulfide bridges in active site of these enzymes may either be not essential or absent.

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The MCL-PHA depolymerase from P. alcaligenes LB19 hydrolyzed the ester bonds of those polymers consisting of medium-chain-length β-hydroxyalkanoates, but it did not hydrolyze the ester linkages of the polymers with short alkyl side chains or hydroxyl groups located in R or ω position on the main carbon chain. A similar observation was also made in the case of the PHO depolymerase from P. fluorescens GK13 and several bacterial lipases.28 Furthermore, increasing the side chain lengths of constituents and the incorporation of C-even β-hydroxyalkanoates into MCLPHA markedly enhanced the degradation of aliphatic MCLPHAs by P. alcaligenes LB19 (Figure 3). Recently, Kim et al.9 demonstrated that a PHPV containing phenyl groups as functional moieties was easily hydrolyzed by the PHPV depolymerase from Xanthomonas sp. JS02, but the copolymer bearing phenoxy groups was degraded much less so. Similarly, in this study, it was found that biodegradation rates of the aromatic PHAs by the MCL-PHA depolymerase from P. alcaligenes LB19 were significantly lower than those of the aliphatic PHAs. The low biodegradability of the aromatic MCL-PHAs might be due to the presence of the bulky aromatic ring structure protecting ester linkages from the enzyme attack. Therefore, it is strongly suggested that the biodegradabilities of polyesters comprised of hydroxyalkanoates can be significantly affected by primarily small differences in microstructures. The substrate specificities for PNP-alkanoates of the MCLPHA depolymerase from P. alcaligenes LB19 were very similar to those of the PHO depolymerase from P. fluorescens GK13.7 The two enzymes commonly hydrolyzed various PNP-alkanoates containing even numbers of carbon atoms (between 2 and 16) in the carbon chain, which are substrates for esterase and/or lipase (Table 4). Comparative results were obtained in PHPV depolymerase from Xanthomonas sp. JS029 and MCL-PHA deploymerase from Pseudomonas sp. RY-1.8 In these cases, the former hardly degraded PNP-palmitate and the latter did not hydrolyze even PNP-octanoate. The purified enzyme from P. alcaligenes LB19 mainly decomposed PHN to the monomeric unit of 3-hydroxynonanoate, and the dimeric and trimeric units of corresponding constituents were also produced. On the basis of these results, it is believed that the enzyme possesses both the endo- and exotypes of depolymerase activity. On the other hand, the PHO depolymerase from P. fluorescens GK13 mainly hydrolyzed PHO to the dimeric form of 3-hydroxyoctanoate.7 In conclusion, the present results suggest that the MCLPHA depolymerase from P. alcaligenes LB19 is an enzyme

Kim et al.

with distinct characteristics, which are different from those of the other MCL-PHA depolymerases reported upon to date. Acknowledgment. This work was supported by a research grant from the Korea Science and Engineering Foundation (Grant No. 1999-2-20200-006-4). References and Notes (1) Madison, L. L.; Huisman, G. W. Microbiol. Mol. Biol. ReV. 1999, 63, 21-53. (2) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451-463. (3) Jendrossek, D. Polym. Degrad. Stab. 1998, 59, 317-325. (4) Foster, L. J. R.; Zervas, S. J.; Lenz, R. W.; Fuller, R. C. Biodegradation 1995, 6, 67-73. (5) Schirmer, A.; Matz, C.; Jendrossek, D. Can. J. Microbiol. 1995, 41 (Suppl. 1), 170-179. (6) Quinteros, R.; Goodwin, S.; Lenz, R. W.; Park, W. H. Int. J. Biol. Macromol. 1995, 25, 135-143. (7) Schirmer, A.; Jendrossek, D.; Schlegel, H. G. Appl. EnViron. Microbiol. 1993, 59, 1220-1227. (8) Kim, H. M.; Ryu, K. E.; Bae, K. S.; Rhee, Y. H. J. Biosci. Bioeng. 2000, 89, 196-198. (9) Kim, H.; Ju, H.-S.; Kim, J. Appl. Microbiol. Biotechnol. 2000, 53, 323-327. (10) Schirmer, A.; Jendrossek, D. J. Bacteriol. 1994, 176, 7065-7073. (11) Kim, Y. B.; Kim, D. Y.; Rhee, Y. H. Macromolecules 1999, 32, 6058-6064. (12) Ramsay, B. A.; Saracovan, I.; Ramsay, J. A.; Marchessault, R. H. J. EnViron. Polym. Degrad. 1994, 2, 1-7. (13) Kim, D. Y.; Kim, Y. B.; Rhee, Y. H. Int. J. Biol. Macromol. 2000, 28, 23-29. (14) Krieg, N. R.; Holt, J. G. Bergey’s manual of systematic bacteriology; The William & Willkins Co.: Baltimore, MD, 1984; Vol. 1. (15) Chung, C. W.; Kim, Y. S.; Kim, Y. B.; Bae, K. S.; Rhee, Y. H. J. Microbiol. Biotechnol. 1999, 9, 847-853. (16) Chun, J. Ph.D. Thesis. University of Newcastle, U.K., 1995. (17) Laemmli, U. K. Nature (London) 1970, 227, 680-685. (18) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. (19) Kita, K.; Ishimaru, K.; Teraoka, M.; Yanase, H.; Kato, N. Appl. EnViron. Microbiol. 1995, 61, 1727-1730. (20) Kim, D. Y.; Kim, Y. B.; Rhee, Y. H. Macromolecules 1998, 31, 4760-4763. (21) Kasuya, K.-I.; Mitomo, H.; Nakahara, M.; Akiba, A.; Kudo, T.; Doi, Y. Biomacromolecules 2000, 1, 194-201. (22) Steinbu¨chel, A.; Fu¨chtenbusch, B. Trends Biotechnol. 1999, 16, 419427. (23) Witholt, B.; Kessler, B. Curr. Opin. Biotechnol. 1999, 10, 279285. (24) Brucato, C. L.; Wong, S. S. Arch. Biochem. Biophys. 1991, 290, 497-502 (25) Mu¨ller, B.; Jendrossek, D. Appl. Microbiol. Biotechnol. 1993, 38, 487-492. (26) Shiraki, M.; Shimada, T.; Tatsumichi, M.; Saito, T. J. EnViron. Polym. Degrad. 1995, 3, 13-21. (27) Sadocco, P.; Nocerino, S.; Dobini-Paglia, E.; Sevres, A.; Elegir, G. J. EnViron. Polym. Degrad. 1997, 5, 57-65. (28) Jaeger, K.-E.; Steinbu¨chel, A.; Jendrossek, D. Appl. EnViron. Microbiol. 1995, 61, 3113-3118.

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