Genetic Analysis and Characterization of Poly(aspartic acid

Abstract. Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing α- and β-amide units and has at least two different types of PAA hyd...
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Biomacromolecules 2003, 4, 80-86

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Genetic Analysis and Characterization of Poly(aspartic acid) Hydrolase-1 from Sphingomonas sp. KT-1 Tomohiro Hiraishi,*,† Mariko Kajiyama,†,‡ Kenji Tabata,† Ichiro Yamato,‡ and Yoshiharu Doi†,§ Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan, and Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8502, Japan Received August 30, 2002; Revised Manuscript Received November 12, 2002

Sphingomonas sp. KT-1 hydrolyzes poly(aspartic acid) (PAA) containing R- and β-amide units and has at least two different types of PAA hydrolases. The PAA hydrolase-1 hydrolyzes selectively β-β amide units in PAA. Molecular cloning of PAA hydrolase-1 from Sphingomonas sp. KT-1 has been carried out to characterize its gene products. Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 and Pseudomonas lemoignei. Site-specific mutation analysis indicates that 176Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue. Introduction Poly(aspartic acid) (PAA), which belongs to a family of synthetic polypeptides, is a biodegradable water-soluble polymer. PAA has several unique characteristics that can be exploited for a range of uses, such as dispersants, detergents, and in biomedical applications.1-5 Because it is a biodegradable water-soluble polymer, PAA has been extensively studied as a replacement for polycarboxylate components. PAA is synthesized by the hydrolysis of polysuccinimide (PSI) prepared by the thermal polymerization of L-aspartic acid (Scheme 1).1,5,6-12 The thermal polymerization of L-aspartic acid with or without phosphate catalyst leads to the formation of a mixture of L- and D-succinimide units in PSI. After hydrolysis of PSI, the resulting PAA is composed of β-amide (70%) and R-amide units (30%) as determined by integration of the methine signals in the 1H NMR spectrum or of the methylene signals in the 13C NMR spectrum.1,13-16 Pivcova´ et al.15 investigated the sequential structure of amide units in the PAA by 13C NMR analysis and concluded that the distribution of R- and β-amide units was random in the PAA sequence. The biodegradation of PAA has been investigated in activated sludge and natural fresh water.1,4,17-20 Freeman et al.18 and Swift et al.4 investigated the biodegradability of PAA polymers synthesized with and without phosphate catalyst and demonstrated that a linear PAA sample was completely degraded in activated sludge. Nakato et al.19 investigated the structural effects on biodegradability of PAA * To whom correspondence should be addressed. Phone: +81-48(467)9403. Fax: +81-48(462)4667. E-mail: [email protected]. † RIKEN Institute. ‡ Science University of Tokyo. § Tokyo Institute of Technology.

Scheme 1. Synthesis of Poly(aspartic acid) (PAA)

in activated sludge. Both the chirality of monomeric units and amide bond structures in PAA did not affect the biodegradability of PAA, while the biodegradability of PAA decreased with an increase in the amount of irregular end groups such as a succinimide end group in PAA. Several techniques such as biochemical oxygen demand (BOD) testing, CO2 evolution testing, TLC, and GPC analyses were used for the investigation of PAA biodegradation in the above articles. To elucidate a detailed mechanism of PAA biodegradation, it is important that isolated bacteria or purified enzymes are used for PAA degradation and that the PAA-degraded products are analyzed by NMR. In our previous studies,20-22 we isolated a PAA-degrading bacterium, Sphingomonas sp. KT-1, from a natural river water, purified a PAA-hydrolyzing enzyme (PAA hydrolase-1), and investigated the biochemical properties of its noble PAA hydrolase-1. Sphingomonas sp. KT-1 has at least two different types of PAA hydrolases,22 and the cell extract hydrolyzed PAA to yield aspartic acid monmer.20 In a previous paper,22 we purified one of the PAA

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Genetic Analysis of Poly(aspartic acid) Hydrolase-1

hydrolases and showed that the PAA hydrolase-1 had some unique characteristics: (i) the N-terminal amino acid sequence of the enzyme had no similarity in the BLASTP search; (ii) the enzymatic degradation products of PAA were aspartic acid oligomers; (iii) the enzyme hydrolyzed specific amide bonds between β-aspartic acid units in PAA. Taking the structure of PAA sequence and the substrate specificity of the PAA hydrolase-1 into consideration, this enzyme plays an important role in PAA degradation in the cell of Sphingomonas sp. KT-1. For more detailed information on PAA hydrolysis, molecular characterization and genetic analysis of the PAA hydrolase-1 are needed. Furthermore, if the enzyme can be purified from recombinant E. coli in good yield, this will be favorable for the characterization and crystallization of the enzyme. In this study, cloning of the PAA hydrolase-1 gene from Sphingomonas sp. KT-1 has been carried out, and the sequence structure and properties of the PAA hydrolase-1 have been reported. To our knowledge, this is the first report on the molecular cloning and characterization of PAA hydrolase from PAA-hydrolyzing microorganisms. Materials and Methods Materials. PAA sample was kindly gifted from Dr. B. Mohr of Polymer Laboratory, BASF, Ludwigshafen, Germany. The PAA was obtained by the hydrolysis of polysuccinimide (PSI) prepared by thermal polymerization of L-aspartic acid with phosphoric acid as a catalyst in the medium for dissolving crystalline aspartic acid at 160-200 °C.22 Other chemicals were purchased from Kanto Chemicals (Tokyo, Japan) or Wako Chemicals (Osaka, Japan). Organism and Growth Conditions. A strain of Sphingomonas sp. KT-1 (JCM10459) isolated from natural river water was grown in medium as described previously.20 Escherichia coli JM109 and BL21(DE3) were used as cloning and expression hosts, respectively. E. coli was usually grown in the Luria-Bertani (LB) broth (1% Bactotrypton, 0.5% Bacto-yeast extract, and 0.5% NaCl, pH 7.0) containing 50 µg/mL of ampicillin. The plasmid vector, pET20b(+), was used for expression of PAA hydrolase-1. Preparation of the plasmid DNA from E. coli and the transformation of E. coli were carried out according to the standard procedures.23 Gene Cloning. PAA hydrolase-1 was purified from Sphingomonas sp. KT-1 as described in our previous study.22 The purified enzyme was digested by CNBr, resulting in the formation of two peptides (PA and PB). The peptides were subjected to amino acid sequence analysis on an Applied Biosystems 473A protein sequencer. The Nterminal amino acid sequences of PA and PB were determined as APAAASKGKAAALPDLKPGAGSFLFTG and SDLVIKYPGLKDAPT, respectively. The N-terminal amino acid sequence of PA was identical with that of mature PAA hydrolase-1. Two degenerated primers were designed for amplification in PCR. Primer N (5′-AARGGIAARGCIGCIGCICTICCIGA-3′) was deduced from the N-terminal amino acid sequence, and primer C (5′-TADTTYATRGGICCIGAITTYCTRCG-3′) was the reversed complement of the

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sequence encoding the digested peptide. The PCR mixture contained 5 µl of 10× PCR buffer, 4 µM deoxynucleoside triphosphate, 4 µM each primer, 1.1 µg of genomic DNA as a template, and 4 U of ExTaq DNA polymerase (TaKaRa, Japan). A DNA thermal cycler (Applied Biosystems Japan Ltd., Japan) was used for amplification of the gene under the following conditions: five cycles of denaturing at 95 °C for 30 s, annealing at 45 °C for 30 s, and extension at 70 °C for 1 min and subsequently 30 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 70 °C for 1 min. An amplified fragment of the expected size was cloned into a pT7Blue T-Vector (Novagen), and its sequence was determined by a dideoxynucleotide chain terminating method using the DNA sequencer CEQ2000 system (Beckman Coulter Inc., U.S.A.). A genomic DNA from Sphingomonas sp. KT-1 was digested completely with the restriction endonuclease Hind III and self-ligated. Two synthetic oligonucleotides, primer 1 (5′-ATGCGGGCTGGTACACGATG-3′) and primer 2 (5′CGTGATCTTGTCGGGCGCGT-3′), served as primers for inverse-PCR amplification. The PCR product was subcloned into pGEM-T Easy (Promega, U.S.A.), and the sequence of the PCR product was determined. Sequence analysis showed that the obtained PCR product was a partial nucleotide because no termination codon was found at the position expected from the molecular weight of PAA hydrolase-1. To determine the complete gene of PAA hydrolase-1, a LA PCR in vitro cloning was performed by using a cloning kit (TaKaRa, Japan) according to the instructions. The genomic DNA was digested completely with the restriction endonuclease BamH I. The DNA fragment was ligated to a cassette oligonucleotide, and used as a PCR template. Primer S1 (5′-ATGCGGGCTGGTACACGATG-3′) and primer S2 (5′-GCATGAGCCAATATGCCCAG-3′) were designed for amplification of the C-terminal region of PAA hydrolase-1 gene. The resultant PCR product was ligated into pGEM-T Easy and the nucleotide sequence of the PCR product was determined. Sequence data were analyzed by the GENETYX programs (Software Development Co., Japan). Database searches were performed with the program FASTA via DDBJ www server. Construction of Plasmid for Expression of PAA Hydrolase-1 Gene. An approximately 950 bp DNA fragment encoding PAA hydrolase-1 was amplified by using genomic DNA from Sphingomonas sp. KT-1 as a template. Two synthetic oligonucleotides, primer 1 (5′-GGTAAAATTCATATGCGCCCCCAG-3′) and primer 2 (5′-GATTGGCAGAGGATCCCCGGCGTCCC-3′), served as primers for PCR amplification. A Nde I restriction site and a BamH I restriction site were introduced at the primer 1 and the primer 2, respectively. PCR conditions were as follows: 30 cycles of denaturation at 96 °C for 30 s, annealing at 70 °C for 30 s, and polymerization at 72 °C for 1.5 min. PCR product was digested by Nde I and BamH I and introduced into pET-20b(+) pretreated with the same restrict enzymes. The resultant plasmid, pPAAI, was introduced into E. coli BL21(DE3) cells, which were then plated onto Luria-Bertani plates containing 100 µg/mL of ampicillin.

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Detection of Recombinant PAA Hydrolase-1. E. coli BL21(DE3) cells harboring pPAAI were cultivated in 1.6 mL of LB medium supplemented with ampicillin (100 µg/ mL) for 3 h at 37 °C, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture medium (final concentration at 0.4 mM). After 3 h cultivation at 30 °C, the cells were harvested by centrifugation at 12 000g at 4 °C for 2 min. To analyze the location of the gene products, the periplasmic fraction was extracted from the cell pellet according to the procedure reported by Koshland and Bostein,24 and the cytoplasmic fraction was prepared by sonication and centrifugation. Detection of the recombinant PAA hydrolase-1 localized in the two fractions, cytoplasm and periplasm, was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the procedure of Laemmli24 with a precision prestained molecular weight calibration kit (Japan Bio-Rad Laboratories, Japan) and immunoblot analysis. Purification of Recombinant PAA Hydrolase-1. E. coli BL21(DE3) cells harboring pPAAI were grown at 37 °C in 1.6 L of LB medium containing 50 µg/mL of ampicillin. When the cell density reached OD ) 0.6-0.8, IPTG was added to the culture medium (final concentration at 0.4 mM). After 3 h cultivation at 30 °C, the cells were harvested by centrifugation at 5000g and 4 °C for 10 min. The collected cells were suspended to 10 mM potassium phosphate buffer (pH 7.0) and disrupted by sonication (20 kHz, 70 W, Tomy Seiko Co. Ltd., Japan). The suspension was ultracentrifugated at 150 000g and 4 °C for 1 h. The resultant supernatant was used for PAA hydrolase-1 purification. All procedures were carried out at 0-4 °C. The soluble fraction was applied to a SP Sepharose HP column (2.6 by 10 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The column was washed with three bed volumes of the same buffer. The enzyme was eluted with a linear gradient from 0 to 250 mM NaCl for 500 mL at 3.0 mL/ min, and the fractions were collected every 6.0 mL. The enzyme fractions were collected, dialyzed against 10 mM potassium phosphate buffer (pH 7.0), and applied to a Hydroxyapatite column (1.6 cm × 10 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The column was washed with three bed volumes of the same buffer. The enzyme was eluted with a linear gradient from 0 to 300 mM potassium phosphate buffer (pH 7.0) for 100 mL at 2.0 mL/ min, and the fractions were collected every 3.0 mL. The enzyme fraction was dialyzed against 10 mM potassium phosphate buffer (pH 7.0) and used as a purified enzyme. PAA-Degrading Assays. PAA-degrading activity of enzyme was analyzed according to the method reported in a previous paper.22 PAA (1.5 mg) was added in 800 µL of 10 mM potassium phosphate buffer (pH 7.0) at 30 °C. The reaction was started by the addition of 10 µL of enzyme solution. After incubation at 30 °C for 30 min, 100 µL of 1 M sodium chloride and 1 mL of ethanol were added in the solution to precipitate unreacted PAA polymers, and the solution was mixed for 1 min. The turbidity of mixed solution was measured at 595 nm. The concentration of unreacted polymers was determined by measuring the turbidity, although the value of turbidity was dependent on molecular

Hiraishi et al.

weight and concentration. One unit of enzyme was defined as the amount of protein required to decrease the value of turbidity at 595 nm by 1 per hour. The PAA products degraded with PAA hydrolase-1 were analyzed by using NMR spectroscopy. The 1H and 13C NMR assignments of reaction products and PAA were made according to the reports of Wolk et al.16 and Matsubara et al.26 Site-Directed Mutagenesis of 176Ser of PAA Hydrolase-1 from Sphingomonas sp. KT-1. Site-directed mutagenesis of the PAA hydrolase-1 gene from Sphingomonas sp. KT-1 was performed according to the literature.27 A pair of oligonucleotide primers, 5′-GCGATCTATGGCCATGCGGCGGGCGGTCAG-3′ and 5′-ATAGGTCGGCACCTTGCTGCCCGTTGCCTTG-3′, was designed in inverted tail-totail directions to amplify the cloning vector together with the target sequence. The pPAAI was used as a template. After the PCR with these primers, amplified linear DNA was selfligated, and the resultant plasmid, pPAAI-S176A, transformed E. coli BL21(DE3) competent cells. S176A mutant was expressed in E. coli and purified as described above. Analytical Procedures. SDS-PAGE was performed according to the procedure of Laemmli25 with a precision prestained molecular weight calibration kit. Protein was stained with Coomassie brilliant blue R250 (KANTO Chemical, Japan). Protein concentrations were determined by the method of Bradford28 with the protein assay kit II (Japan Bio-Rad Laboratories, Japan) and bovine serum albumin (BSA) was used as a standard. Results and Discussion Gene Analysis of PAA Hydrolase-1 from Sphingomonas sp. KT-1. Figure 1 shows the DNA sequence and deduced amino acid sequence of PAA hydrolase-1 from Sphingomonas by cloning procedures. As a result, the PAA hydrolase-1 gene was composed of 942 bp, revealing that the open reading frame starts from the putative initiation codon ATG at nucleotide 1, located 10 bp downstream of the putative Shine-Dalgarno (S/D) sequence. The encoded polypeptide was a preprotein of 314 amino acids with a predicted molecular weight of 34 087 Da. When the deduced amino acid sequence of PAA hydrolase-1 was compared with the mature PAA hydrolase-1 from Sphingomonas sp. KT-1, the 35 amino acid polypeptide from N-terminal was found to be a signal peptide. The molecular weight deduced from PAA hydrolase-1 gene was 30 812 Da, which was in agreement with the value (30 kDa) determined by SDS-PAGE (Figure 3, lane 1). The gene analysis showed that PAA hydrolase-1 conserved the lipase box (Gly-Xaa-176Ser-Xaa-Gly), which functions as an active center in well-known serine hydrolases. We demonstrated that phenylmethane sulfonyl fluoride (PMSF) or diisopropyl fluorophosphates (DFP) strongly inhibited the activity of PAA hydrolase-1.22 The presence of the lipase box is consistent with the result of effect of inhibitor on PAA hydrolase-1 activity. Homology of the deduced amino acid sequence of PAA hydrolase-1 was searched by using FASTA. The result of FASTA showed that the amino acid sequence of PAA

Genetic Analysis of Poly(aspartic acid) Hydrolase-1

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Figure 1. The DNA and deduced amino acid sequences of PAA hydrolase-1 from Sphingomonas sp. KT-1. A putative ribosome-binding (ShineDalgarno [S/D]) site is boxed. Amino acids confirmed by Edman degradation are underlined, and the processing site of the PAA hydrolase-1 precursor is marked by a vertical arrow.

hydrolase-1 was comparatively similar to those of catalytic domains in the poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 (PhaZAfaAE122) (26.5% identity, 257 aa) and from Pseudomonas lemoignei (PhaZ2Ple) (25.8% identity, 244 aa) (Figure 2). In addition, the multialignment of deduced amino acid sequence of PAA hydrolase-1 suggests that PAA hydrolase-1 may conserve 244 Asp as a one of the residues composing a catalytic triad and 89His as an oxyanion hole and that the sequential order of the catalytic triad amino acids in PAA hydrolase-1 may be histidine(oxyanion hole)-serine-aspartate-histidine though the last histidine was not found by the gene analysis. In the

majority of PHB depolymerases, the sequential order of the catalytic triad amino acids is histidine(oxyanion hole)-serineaspartate-histidine. These findings suggest that the structure of PAA hydrolase-1 may be similar to those of the catalytic domain of PHB depolymerases. Characterization of Wild-type PAA Hydrolase-1 from Recombinant E. coli. The PAA hydrolase-1 gene was amplified and introduced into expression vector pET-20b(+), and the resultant plasmid (pPAAI) was introduced into E. coli BL21(DE3) cells. The recombinant E. coli was cultured until mid-log phase at 37 °C. After the addition of 0.4 mM IPTG and further cultivation at 30 °C for 3 h, a

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Figure 2. Multialignment of the putative amino acids of PAA hydrolase-1 from Sphingomonas sp. KT-1 with those of PHB depolymerases. The sequences of the PAA hydrolase-1 from Sphingomonas sp. KT-1 and the PHB depolymerases from A. faecalis AE122 (PhaZAfaAE122) and P. lemoignei (PhaZ2Ple) are shown.

Figure 3. SDS-PAGE analysis of purified PAA hydrolases: (M) molecular mass standard; (1) wild-type of PAA hydrolase-1 purified from Sphingomonas sp. KT-1; (2) wild-type of PAA hydrolase-1 purified from recombinant E. coli; (3) S176A mutant of PAA hydrolase-1 from recombinant E. coli.

high PAA-degrading activity was observed in the soluble protein fraction. The soluble protein fraction prepared from the cells grown in 1.6 L cultures was applied onto a SP Sepharose HP column followed by a Hydroxyapatite column. SDS-PAGE analysis revealed that the collected active fraction was electrophoretically homogeneous (Figure 3, lane 2). The specific activity of wild-type enzyme (135 U/mg) from recombinant E. coli was almost the same as that of the enzyme purified from Sphingomonas sp. KT-1 (140 U/mg) (Table 1). The structure of degraded products of PAA polymer with the enzyme from recombinant E. coli was characterized by

using NMR spectroscopy as reported in a previous paper.22 The fraction of β-bonded monomeric units in a main chain of PAA polymer was decreased from 70 to 47 mol % after enzymatic hydrolysis for 24 h, while the fraction (30 mol %) of R-bonded monomeric units in main chain remained almost unchanged in the intensities of NMR resonances during the course of enzymatic hydrolysis (Figure 4), suggesting that the PAA hydrolase-1 hydrolyzes selectively the amide bonds of β-aspartic acids in PAA. This degradation behavior of PAA with wild-type enzyme from recombinant E. coli was the same as that with wild-type enzyme from Sphingomonas sp. KT-1 as reported in a previous paper.22 These results demonstrated that the enzyme from recombinant E. coli possessed the same activity and substrate specificity in comparison with the wild-type enzyme from Sphingomonas sp. KT-1. When the purified enzyme from recombinant E. coli was subjected to Edman degradation, an N-terminal amino acid sequence of PAA hydrolase-1 was identified as SKGKAAALPDLKPGAGSFLF (Table 1). This result indicates that five amino acids from N-terminal are unnecessary on PAA hydrolase-1 activity. Two reasons for the digestion of the N-terminal amino acids may be proposed as follows: First, the signal peptide of PAA hydrolase-1 was incorrectly digested by a signal peptidase in E. coli cells during secretion of PAA hydrolase-1. Second, the N-terminal amino acids of PAA hydrolase-1 were hydrolyzed by a protease produced in E. coli after secretion of PAA hydrolase-1. To identify whether the PAA hydrolase-1 was effectively secreted in E. coli, the localization of PAA hydrolase-1 in E. coli cells was analyzed. The cytoplasmic and periplasmic

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Genetic Analysis of Poly(aspartic acid) Hydrolase-1

Table 1. Specific Activities and N-Terminal Amino Acid Sequences of PAA Hydrolase-1 from Sphingomonas sp. KT-1 and Recombinant E. coli BL21(DE3) enzyme

specific activity (U/mg)

N-terminal amino acid sequence

wild-type from Sphingomonas sp. KT-1 wild-type from E. coli BL21(DE3) S176A mutant from E. coli BL21(DE3)

140 135 0

APAAASKGKAAALPDLKPG SKGKAAALPDLKPG SKGKAAALPDLKPG

Figure 5. Chemical structures of β-β diad sequence of (a) poly(aspartic acid) (PAA) and (b) poly(3-hydroxybutyric acid) (PHB). The hydrolysis sites of PAA and PHB with enzymes are marked by a vertical arrow.

Figure 4. 13C NMR spectra of carbonyl carbon in PAA and degraded products with PAA hydrolase-1 purified from recombinant E. coli: (a) PAA; (b) degraded products after 24 h of enzymatic hydrolysis.

fractions were prepared by using osmotic shock procedure and applied to SDS-PAGE and immunoblot analysis. A specific band of PAA hydrolase-1 with apparently the same molecular mass as that of authentic PAA hydrolase-1 was identified in the periplasmic fraction (data not shown). On the other hand, two specific bands of PAA hydrolase-1 were observed in the cytoplasmic fraction. One was apparently the same molecular mass as a specific band of authentic PAA hydrolase-1, while the molecular mass of another band was larger than that of authentic PAA hydrolase-1. In addition, the analysis of N-terminal amino acid sequence of PAA hydrolase-1 from recombinant E. coli shows that PAA hydrolase-1 may be hydrolyzed by a protease produced in E. coli after expression of PAA hydrolase-1. These results suggest that the structure of the PAA hydrolase-1 gene may not be suitable for the secretory expression system in E. coli and that the regulation mechanism of expression and secretion of PAA hydrolase-1 in Sphingomonas is different from that in E. coli. For an effective secretion of PAA hydrolase1, it is necessary to construct a suitable system for gene expression of PAA hydrolase-1 in E. coli by replacement of its leader sequence with another.

Purification and Characterization of S176A Mutant. To examine whether the 176Ser amino acid residue in the lipase box of PAA hydrolase-1 plays as an active center, we carried out site-specific mutation of serine to alanine at the position of 176. The mutant enzyme showed the same binding properties during the purification procedure of protein as the corresponding wild-type enzyme from recombinant E. coli, and the relative molecular mass of the S176A protein in SDS-PAGE was consistent with that of wild-type protein from recombinant E. coli (Figure 3, lanes 2 and 3). Therefore, we have concluded that the mutation did not affect the biochemical properties of PAA hydrolase-1. However, when the activity of the purified S176A mutant enzyme was measured, the enzyme was completely inactive for PAA hydrolysis (Table 1), indicating that 176Ser of PAA hydrolase-1 is essential as an active residue for PAA hydrolysis. In a previous paper,22 we demonstrated that the PAA hydrolase-1 hydrolyzed selectively the amide bonds between β-aspartic acid units in PAA. The chemical structure of diad linkage of β,β-aspartic acids in PAA resembles that of diad linkage of 3-hydroxybutyric acids in PHB, which are also connected at β-position, as shown in Figure 5. In addition, the deduced amino acid sequence of PAA hydrolase-1 has a homology to those of the catalytic domain in PHB depolymerases, suggesting that the PAA hydrolase-1 and PHB depolymerases may have the same evolutionary origin. Conclusions This paper has reported the genetic analysis and characterization of poly(aspartic acid) (PAA) hydrolase-1 from Sphingomonas sp. KT-1 (JCM10459). Genetic analysis shows that the deduced amino acid sequence of PAA hydrolase-1 has a similarity with those of the catalytic domain of poly(3-hydroxybutyric acid) (PHB) depolymerases from Alcaligenes faecalis AE122 (26.5% identity, 257 aa) and from Pseudomonas lemoignei (25.8% identity, 244 aa). When PAA hydrolase-1 was expressed in E. coli BL21(DE3), the five amino acids from N-terminal of recombinant PAA

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hydrolase-1 were digested. However, the recombinant PAA hydrolase-1 possessed the same activity and substrate specificity in comparison with the wild-type enzyme from Sphingomonas sp. KT-1, indicating that five amino acids from N-terminal are unnecessary on PAA hydrolase-1 activity. Site-specific mutation analysis of the PAA hydrolase-1 indicates that 176Ser is a part of a strictly conserved pentapeptide sequence (Gly-Xaa-Ser-Xaa-Gly), which is the lipase box, and plays as an active residue. Acknowledgment. We gratefully acknowledge Dr. Bernhard Mohr of BASF, Germany, for supplying PAA sample. This research was supported by a grant for Ecomolecular Science Research to RIKEN Institute and SORST (Solution Oriented Research for Science and Technology) grant from the Japan Science and Technology Corporation (JST). References and Notes (1) Roweton, S.; Huang, S. J.; Swift, G. J. EnViron. Polym. Degrad. 1997, 5, 175. (2) Amass, W.; Amass, A.; Tighe, B. Polym. Int. 1998, 47, 89. (3) Karlsson, S.; Albertsson, A. Polym. Eng. Sci. 1998, 38, 1251. (4) Swift, G.; Freeman, M. B.; Paik, Y. H.; Simon, E.; Wolk, S.; Yocom, K. M. Macromol. Symp. 1997, 123, 195. (5) Nakato, T.; Kusuno, A.; Kakuchi, T. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 117. (6) Vegotsky, A.; Harada, K.; Fox, S. W. J. Am. Chem. Soc. 1958, 80, 3361. (7) Kokufuta, E.; Suzuki, S.; Harada, K. Bull. Chem. Soc. Jpn. 1978, 51, 1555. (8) Tomida, M.; Nakato, T.; Kuramachi, M.; Shibata, M.; Matsunami, S.; Kakuchi, T. Polymer 1996, 37, 4435.

Hiraishi et al. (9) Kovacs, J.; Kovacs, H. N.; Ko¨nyves, I.; Csa´sza´r, J.; Vajda, T.; Mix, H. J. Org. Chem. 1961, 26, 1084. (10) Schwamborn, M. Polym. Degrad. Stab. 1998, 59, 39. (11) Low, K. C.; Wheeler, A. P.; Koskan, L. P. AdV. Chem. Ser. 1996, 248, 99. (12) Neri, P.; Antoni, G.; Benvenuti, F.; Cocola, F.; Gazzei, G. J. Med. Chem. 1973, 16, 893. (13) Matsuyama, M.; Kokufuta, E.; Kusumi, T.; Harada, K. Macromolecules 1980, 13, 196. (14) Pivcova´, H.; Saudek, V.; Drobnı´k, J.; Vlasa´k, J. Biopolymer 1981, 20, 1605. (15) Pivcova´, H.; Saudek, V.; Drobnı´k, J. Polymer 1982, 23, 1237. (16) Wolk, S. K.; Swift, G.; Paik, Y. H.; Yocom, K. M.; Smith, R. L.; Simon, E. S. Macromolecules 1994, 27, 7613. (17) Alford, D. D.; Wheeler, A. P.; Pettigrew, C. A. J. EnViron. Polym. Degrad. 1994, 2, 225. (18) Freeman, M. B.; Paik, Y. H.; Swift, G.; Wilczynski, R.; Wolk, S. K.; Yocom, K. M. Polym. Repr. 1994, 35, 423. (19) Nakato, T.; Yoshitake, M.; Matsubara, K.; Tomida, M.; Kakuchi, T. Macromolecules 1998, 31, 2107. (20) Tabata, K.; Kasuya, K.; Abe, H.; Masuda, K.; Doi, Y. Appl. EnViron. Microbiol. 1999, 65, 4268. (21) Tabata, K.; Abe, H.; Doi, Y. Biomacromolecules 2000, 1, 157. (22) Tabata, K.; Kajiyama, M.; Hiraishi, T.; Abe, H.; Doi, Y. Biomacromolecules 2001, 2, 1155. (23) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: a Laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (24) Koshland, D.; Botstein, D. Cell 1980, 20, 749. (25) Leammli, U. K. Nature 1970, 227, 680. (26) Matsubara, K.; Nakato, T.; Tomida, M. Macromolecules 1998, 31, 1466. (27) Imai, Y.; Matsushima, Y.; Sugimura, T.; Terada, M. Nucleic Acids Res. 1991, 19, 2785. (28) Bradford, M. M. Anal. Biochem. 1976, 72, 248.

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