Biochemical and Molecular Characterization of Poly (aspartic acid

Biomacromolecules 2003, 4, 1285-1292

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Biochemical and Molecular Characterization of Poly(aspartic acid) Hydrolase-2 from Sphingomonas sp. KT-1 Tomohiro Hiraishi,*,† Mariko Kajiyama,†,‡ Kenji Tabata,† Hideki Abe,† 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 March 20, 2003; Revised Manuscript Received July 1, 2003

Poly(aspartic acid) (PAA) hydrolase-2 was purified from crude soluble cellular extracts of Sphingomonas sp. KT-1 (JCM10459) and characterized to elucidate the mechanism of R,β-poly(D,L-aspartic acid) (tPAA) biodegradation. The molecular mass of PAA hydrolase-2 was 42 kDa, and the isoelectric point was 9.6. The optimum values of pH and temperature for the hydrolysis of R-di(L-aspartic acid) by PAA hydrolase-2 were 7.0 and 55 °C, respectively. The effect of inhibitors on the hydrolysis of R-di(L-aspartic acid) showed that the activity of PAA hydrolase-2 was significantly inhibited by EDTA. Thermally synthesized tPAA was hydrolyzed in the presence of two enzymes, PAA hydrolase-1 and PAA hydrolase-2, to generate aspartic acid. The PAA hydrolase-2 was capable of hydrolyzing R-poly(L-aspartic acid) of high molecular weights but had limited activity for tPAA. These results lead us to propose the following mechanism. First, PAA hydrolase-1 hydrolyzes tPAA to yield oligo(aspartic acid) via an endo-mode cleavage, and subsequently, PAA hydrolase-2 hydrolyzes the resultant oligo(aspartic acid) to yield aspartic acid. Analysis of hydrolyzed products from R- and β-penta(L-aspartic acid) revealed that PAA hydrolase-2 catalyzed the exo-mode hydrolysis of R- and β-penta (L-aspartic acid). The gene encoding PAA hydrolase-2 from Sphingomonas sp. KT-1 was cloned, and genetic analysis showed that the deduced amino acid sequence of PAA hydrolase-2 is similar to a putative peptidase, which belongs to the M20/M25/M40 family of proteins, from Caulobacter crescentus CB15. Introduction Polyanionic water-soluble polymers are used as dispersants, detergent builders, and paper additives.1,2 One of the most widely used water-soluble polymers for these applications is poly(acrylic acid), however poly(acrylic acid) is not biodegradable and accumulates in the environment. Because of the potential environmental damage associated with the use of poly(acrylic acid), research on biodegradable watersoluble polymers such as poly(malic acid), poly(glutamic acid), and poly(aspartic acid) (PAA) has become an important research topic.3-7 Poly(malic acid) is synthesized by both chemical process and from certain microorganisms, and it shows excellent biodegradability and biocompatibility.8 However, production costs of poly(malic acid) are fairly high and, because the malic acid units are connected via ester linkage, it is unstable even in slightly alkaline media. Therefore, poly(malic acid) may have limited applications. On the other hand, the monomers of poly(glutamic acid) and PAA are connected by amide links and are thus more stable in alkaline solution than poly(malic acid). Because the * 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.

content of carboxyl groups per molecular weight in poly(glutamic acid) is lower than that in PAA, the polyanionic ability of poly(glutamic acid) is inferior to that of PAA.9 In addition, thermal synthesized R,β-poly(D,L-aspartic acid) (tPAA) has been chemically produced on a large scale by the hydrolysis of polysuccinimide (PSI) synthesized via the thermal polymerization of L-aspartic acid (structure 1).4,10,11,14,19,20 Therefore, PAA has been extensively studied as a replacement for commercial polycarboxylate components.12-18

It was shown previously that complex microbial communities and relatively high C:N ratios optimized mineralization of tPAA.22 To elucidate a detailed mechanism of tPAA biodegradation, it is important that isolated microorganisms or purified enzymes are used for the research of tPAA degradation. Until now, there have been only a few papers regarding the tPAA biodegradation by isolated tPAAdegrading bacteria.23-25 In our previous studies,23,24 we

10.1021/bm034085i CCC: $25.00 © 2003 American Chemical Society Published on Web 08/08/2003

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isolated two types of PAA-degrading bacteria (Sphingomonas sp. KT-1 (JCM10459) and Pedobacter sp. KP-2 (JCM10638)) and investigated tPAA degradation by these bacteria in detail. Sphingomonas sp. KT-1 could completely degrade tPAA of low-molecular-weights below 5000, whereas Pedobacter sp. KP-2 could degrade high-molecular-weight tPAA to release oligo(aspartic acid) (OAA) as a product. A mixed culture of Sphingomonas sp. KT-1 with Pedobacter sp. KP-2 resulted in a complete degradation of high-molecular-weight tPAA. These results suggest that at least two kinds of PAAdegrading enzymes may participate in tPAA degradation. Moreover, the cell extract of Sphingomonas sp. KT-1 could degrade high-molecular-weight tPAA over 5000 to yield aspartic acid, suggesting that Sphingomonas sp. KT-1 has unusual enzymes capable of hydrolyzing β-amide linkages. These findings led us to purify one of the PAA-hydrolyzing enzymes (PAA hydrolase-1) from Sphingomonas sp. KT-1 and subsequent cloning of the gene for this enzyme.26, 27 PAA hydrolase-1 enzyme hydrolyzed specific bonds between β,β-aspartic acid units in tPAA to produce oligo(aspartic acid) (OAA); however, this enzyme cannot degrade OAA to aspartic acid.27 These findings strongly suggested that another type of enzyme (PAA hydrolase-2), which hydrolyzes OAA to aspartic acid, synergistically participates to degrade the tPAA to aspartic acid in Sphingomonas sp. KT-1. Therefore, we have carried out the purification and characterization of PAA hydrolase-2. In the present study, we report the purification of PAA hydrolase-2 from Sphingomonas sp. KT-1 and the biochemical properties of this enzyme. In addition, the molecular cloning of PAA hydrolase-2 gene has been carried out, and the tPAA hydrolysis mechanism in the cell of Sphingomonas sp. KT-1 is discussed. Materials and Methods Materials. The R,β-poly(D,L-aspartic acid) (tPAA) sample was a gift from Dr. B. Mohr of Polymer Laboratory, BASF, Ludwigshafen, Germany. The tPAA was obtained by hydrolyzing polysuccinimide (PSI) prepared by thermal polymerization of L-aspartic acid with phosphoric acid as a catalyst at 160-200 °C. The molecular weight of the polymer was determined by a GPC system (Millennium 486 system, Waters) with columns of Shodex OHpak SB-804 (Showadenko K. K., Japan) and Superdex peptide (Amersham Bioscience). Poly(acrylic acid) standard samples (Polymer Science) were used for calibration. Sodium nitrate (0.4 M) was used for the mobile phase at a flow rate of 0.5 mL/min with a refractive index detector to detect samples. R-Di(Laspartic acid), R-penta(L-aspartic acid), and R-poly(L-aspartic acid) were purchased from Sigma (Japan). β-Penta(L-aspartic acid) was purchased from Sigma Genosis (Japan). Other chemicals were purchased from Kanto Chemicals (Tokyo, Japan) or Wako Chemicals (Osaka, Japan). Organism and Growth Conditions. Sphingomonas sp. KT-1 (JCM10459) was grown in mineral medium at 25 °C as described.26 Escherichia coli JM109 was used as a cloning host and was grown in Luria-Bertani (LB) broth (1% Bactotryptone, 0.5% Bacto-yeast extract and 0.5% NaCl, pH 7.0)

Hiraishi et al.

containing 50 µg/mL ampicillin. Preparation of plasmid DNA from E. coli and the transformation of E. coli were carried out according to standard procedures.30 Purification of PAA Hydrolase-2 from Sphingomonas sp. KT-1. After cultivation of Sphingomonas sp. KT-1 at 25 °C for 24 h in 6.0 L of mineral medium containing L-aspartic acid as a carbon source, cells were collected by centrifugation (5000 g, 4 °C for 15 min) and washed with 10 mM sodium phosphate buffer (pH 7.0). The pellet was suspended in the same buffer and was sonicated on ice for 30 min with a 20 kHz ultrasonic oscillator (Tomy Seiko Co. Ltd., Japan). The disrupted cells were centrifuged at 150 000 g for 1 h at 4 °C, and the supernatant (soluble fraction) was subjected to further purification. All purification steps were performed at 0-4 °C. The soluble fraction was applied to a SP Sepharose HP column (2.6 by 10 cm) equilibrated with 10 mM sodium phosphate buffer (pH 7.0), and 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 5.0 mL/min, and the fractions were collected over 2 min intervals. The enzyme activity was eluted at approximately 150 mM NaCl. The enzyme fractions were collected, dialyzed against 10 mM sodium phosphate buffer (pH 7.0), and applied to a Hydroxyapatite column (1.6 by 10 cm) equilibrated with 10 mM sodium 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 10 to 250 mM sodium phosphate buffer (pH 7.0) for 100 mL at 2.0 mL/min, and the fractions were collected over 6 min intervals. The enzyme activity was eluted at approximately 150 mM sodium phosphate. The enzyme fraction was dialyzed against 10 mM sodium phosphate buffer (pH 7.0) and used as a purified enzyme. Analysis of r-Di(L-aspartic acid) Hydrolysis by PAA Hydrolase-2. The activity of PAA hydrolase-2 was assayed by the hydrolysis of R-di(L-aspartic acid). First, 5 mM of R-di(L-aspartic acid) was prepared in 100 µL of 10 mM sodium phosphate buffer (pH 7.0) at 30 °C and the reaction was started by the addition of enzyme solution (0.75 µg/ mL). The reaction was incubated at 30 °C for 1 h, and the amount of generated L-aspartic acid was determined according to the literature.31 The reaction mixture (10 µL) was added to the solution containing 18 µg/mL glutamicoxaloacetic transaminase, 40 µg/mL malate dehydrogenase, 2 mM R-ketoglutarate, and 0.2 mM NADH in 300 µL of 10 mM sodium phosphate buffer (pH 7.0) followed by measurement of the disappearance of absorbance at 340 nm derived from the decrease of NADH. One unit of enzyme was defined as the amount of protein required to degrade 1 µmol of R-di(L-aspartic acid) per min. Analysis of Degradation Products of tPAA by PAA Hydrolase-2. The hydrolysis of R-penta(L-aspartic acid) and β-penta(L-aspartic acid) by purified PAA hydrolase-2 from Sphingomonas sp. KT-1 was carried out at 30 °C in 0.4 mL of 10 mM sodium phosphate buffer (pH 7.0), in which the reaction was started by the addition of PAA hydrolase-2 (100 µg/mL). The products of the hydrolysis were analyzed by high performance liquid chromatography (HPLC). Briefly,

Characterization of Poly(aspartic acid) Hydrolase-2

50 µL of samples of the reaction mixture were injected into the stainless steel column containing Mightysil RP-18 Aqua 250-46 at 40 °C, and gradient of distilled water (pH 2.5, adjusted by the addition of HCl solution) to 5% acetonitrile was carried out for 10 min with a flow rate of 1.0 mL/min using Shimadzu LC-9A HPLC system with a gradient controller. Products were detected at 210 nm with an SPD10A UV spectrophotometric detector. For the separation of the R-forms of aspartic acids, R-tri(L-aspartic acid), R-tetra(L-aspartic acid), and R-penta(L-aspartic acid) were detected at 210 nm and eluted at 3.4, 4.0, and 4.7 min, respectively. However, L-aspartic acid and R-di(L-aspartic acid) were eluted at 3.0 min and could not be separated from each other in this method. On the other hand, the β-forms of aspartic acids [L-aspartic acid, β-di(L-aspartic acid), β-tri(L-aspartic acid), β-tetra(L-aspartic acid), and β-penta(L-aspartic acid)] could be separated by the above method and eluted at 3.0, 3.4, 3.8, 4.2, and 4.8 min, respectively. Gel Permeation Chromatography (GPC) Analysis of Hydrolyzed Product of PAA by PAA Hydrolase-2. The hydrolysis behavior of R-poly(L-aspartic acid), tPAA, and oligo(aspartic acid) (OAA) with PAA hydrolase-2 was also investigated by the GPC method. Each substrate was incubated with PAA hydrolase-2 in 10 mM sodium borate buffer (pH 8.0). After enzymatic treatment, 200 µL of the reaction mixture was subjected to the GPC system with columns of Shodex OHpak SB-804 and Superdex peptide at 30 °C. Sodium nitrate (0.4 M) was used for the mobile phase, and the flow rate was 0.5 mL/min. A refractive index detector was used to detect products, and poly(acrylic acid) standard samples were used for calibration. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed according to the procedure of Laemmli32 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 Bradford33 with the protein assay kit II (Japan Bio-Rad Laboratories, Japan), and bovine serum albumin (BSA) was used as a standard. Isoelectric Focusing (IEF) of PAA Hydrolase-2. IEF was performed by Multiphor II system (Amersham Bioscience). Immnobiline DryStrip (pH 3-10.5) (Amersham Bioscience) and Broad pI kit (pH 3.5-9.3) (Amersham Bioscience) were used as gel and marker, respectively. N-Terminal Amino Acid Sequence of PAA Hydrolase2. For N-terminal amino acid sequencing, the protein separated by SDS-PAGE was transferred electrophoretically onto Immobilon-P transfer membrane and visualized by staining with Coomassie brilliant blue R-250. The blotted protein was cut out from the membrane and subjected to N-terminal amino acid sequence analysis on an Applied Biosystems 473A protein sequencer. Gene Cloning. The N-terminal amino acid sequence of the mature PAA hydrolase-2 was determined as APKAKPEAVLKTKGYEAAVKILDRDHDRMVDEIIKLTEIPAP. To amplify the DNA fragment corresponding to the determined amino acid sequence, two degenerate primers were designed to amplify the gene by PCR. Primer N (5′-

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CCIAARGCIAARCCIGARGCIGT-3′) was deduced from the amino acid sequence PKAKPEAV, and primer C (5′TTDATIATYTCRTCIACCATIC-3′) was the reversed complement of the sequence encoding amino acids RMVDEIIK. The PCR mixture contained 5 µL of 10 × PCR buffer, 0.4 mM deoxynucleoside triphosphate, 4 µM of 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: 5 cycles of denaturing at 95 °C for 30 s, annealing at 35 °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 45 °C for 30 s, and extension at 70 °C for 1 min. An amplified fragment of the expected size was cloned into a pGEM-T Easy (Promega, U.S.A.), and the internal nucleotide sequence was determined. To obtain the whole PAA hydrolase-2 gene, in vitro cloning was performed by using the LA PCR in vitro cloning kit (TaKaRa, Japan) according to the instructions. Genomic DNA was digested completely with the restriction endonucleases as PstI, SalI, or BamHI. The DNA fragments were ligated to cassette oligonucleotides and used as PCR templates. To amplify the upstream region of the internal sequence of the determined amino acids (LKTKGYEAAVKILDRDH), primer S1 (5′-GGTCGCGGTCGAGGATCTTGACC-3′) and primer S2 (5′-GCCGCTTCATAGCCCTTGGTCTTCA-3′) were designed. For the downstream region of the internal sequence of the determined amino acids, primer S3 (5′-TGAAGACCAAGGGCTATGAAGCGGC-3′) and primer S4 (5′-GGTCAAGATCCTCGACCGCGACC3′) were designed. After LA PCR, the resultant PCR products were ligated into pGEM-T Easy. The nucleotide sequence was determined by a dideoxynucleotide chain terminating method using a DNA CEQ2000 sequencer (Beckman Coulter Inc., U.S.A.). Sequence data were analyzed by the GENETYX programs (Software Development Co., Tokyo, Japan). Sequence analysis showed that the obtained PCR products were partial nucleotide sequences because no termination codon could be found at the position expected from the molecular weight of the mature PAA hydrolase-2. To determine the complete gene of PAA hydrolase-2, genomic Southern hybridization and colony hybridization were carried out. Two primers were designed on the basis of a partial nucleotide sequence of PAA hydrolase-2 to prepare DNA probes for screening a whole PAA hydrolase-2 gene. The sequences of the primers were as follows: primer 1 (5′-GAAGACCAAGGGCTATGAAGCGGCG-3′) and primer 2 (5′-TGTAGAAATCGACGACCGTCTGGCTCATC3′). PCR was performed to amplify the DNA fragment by using genomic DNA as a template. The amplified DNA was labeled by a digoxigenin (DIG) DNA labeling kit (Roche Diagnostics, Germany). Genomic DNA was digested completely with the restriction endonuclease as ClaI. The DNA was separated by electrophoresis, blotted onto a positively charged nylon membrane, and hybridized with a DIG-labeled DNA probe. Positive DNA fragments (ca. 5 kbp) were subcloned into the pBluescript II KS(+) (STRATAGENE) pretreated with ClaI, and the resultant plasmid was trans-

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Table 1. Purification of PAA Hydrolase-2 from Sphingomonas sp. KT-1

soluble fraction SP Sepharose HP hydroxyapatite

total activity (U)

specific activity (mU/mg)

total protein (mg)

yield (%)

2.1 1.8 1.4

0.9 180 700

2400 10 2.0

100 86 64

formed into E. coli JM109. Colony hybridization of genomic sublibraries with a DIG-labeled DNA probe was performed. By using the ClaI DNA fragment as a template, primer walking was performed to obtain the nucleotide sequence of the entire PAA hydrolase-2 gene. Sequence data were analyzed by the GENETYX program, and database searches were performed with the program FASTA via DDBJ www server. Nucleotide Sequence Accession Number. The nucleotide sequence data reported in this paper have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB112787. Results and Discussion Purification and Properties of PAA Hydrolase-2 from Sphingomonas sp. KT-1. PAA hydrolase-2 was purified to homogeneity from Sphingomonas sp. KT-1 as described in the Materials and Methods section. Details of the purification of PAA hydrolase-2 are listed in Table 1. The enzyme was purified 778-fold with an overall yield of 64%. SDS-PAGE analysis of the purified enzyme fraction revealed a single polypeptide band at 42 kDa (data not shown). The N-terminal amino acid sequence of the purified enzyme was determined as APKAKPEAVLKTKGYEAAVKILDRDHDRMVDEIIKLTEIPAP by automated Edman degradation. BLAST search analysis revealed that the N-terminal amino acid sequence of PAA hydrolase-2 is similar to a putative peptidase, which belongs to M20/M25/M40 family of proteins, from Caulobacter crescentus CB15.34 The isoelectric point of PAA hydrolase-2 was determined as 9.6 by isoelectric focusing, and the pH dependency on the hydrolysis of R-di(L-aspartic acid) with PAA hydrolase-2 was determined in 10 mM sodium acetate buffer (pH 4-5), 10 mM sodium dihydrogen phosphate buffer (pH 5-8), 10 mM sodium borate buffer (pH 8-10), and 10 mM sodium carbonate buffer (pH 10-11). The hydrolysis activity of PAA hydrolase-2 for R-di(L-aspartic acid) was found between pH 5 and 11 with an optimum pH of 7.0 for hydrolysis of R-di(L-aspartic acid). As shown in Figure 1, the optimum temperature for hydrolysis was 55 °C. The activity of PAA hydrolase-2 on the hydrolysis of R-di(L-aspartic acid) remained unchanged after 3 h of incubation at 50 °C. The properties (relatively high optimum temperature and high thermostability) of PAA hydrolase-2 are interesting because the optimum temperature for the cell growth of Sphingomonas sp. KT-1 is 28 °C. The effect of protease inhibitors on the hydrolysis of R-di(L-aspartic acid) with the purified enzyme was investigated, and the results are listed in Table 2. The hydrolysis of R-di-

Figure 1. Effect of temperature on the hydrolysis activity of PAA hydrolase-2 for R-di(L-aspartic acid). Reactions of the R-di(L-aspartic acid) hydrolysis were performed at different temperatures in 10 mM sodium phosphate buffer (pH 7.0). The activity of PAA hydrolase-2 on the R-di(L-aspartic acid) hydrolysis at 30 °C in 10 mM sodium phosphate buffer (pH 7.0) was defined as 100%. Table 2. Effect of Protease Inhibitors on theActivity of PAA Hydrolase-2a inhibitor

relative activity (%)

none EDTA (1 mM) DFP (3 mM) PMSF (10 mM) pepstatin (8 µM) aprotinin (1 µM) E64 (0.1 mM)

100 16 14 24 63 92 66

a PAA hydrolase-2 (0.4 µM) was incubated with each inhibitor for 15 min at 30 °C. After incubation, R-di(L-aspartic acid) degrading activities were measured. EDTA, ethylenediaminetetraacetic acid; DFP, diisopropylfluorophosphates; PMSF, phenylmethane sulfonyl fluoride; E64, N-[N(L-3-trans-carboxirane-2-carbony l)-L-leucyl]agmatine.

(L-aspartic acid) was significantly inhibited by the metalloprotease inhibitor EDTA, suggesting that PAA hydrolase-2 may require metal ion(s) for activity. In addition, serinetype protease inhibitors such as diisopropyl fluorophosphate (DFP) and phenylmethane sulfonyl fluoride (PMSF) also inhibited the hydrolysis activity of the purified enzyme. Enzymatic Hydrolysis of r-Poly(L-aspartic acid), tPAA, and OAA. The structure-biodegradability relationship for thermal synthesized R,β-poly(D,L-aspartic acid) (tPAA) has been investigated in active sludge.12,14-18,21 Recently, Nakato et al. found that both the chirality (L or D) of monomeric units and amide bond structure (R or β) in tPAA had no effect on the biodegradability of tPAA, whereas the biodegradability of tPAA decreased with an increase of the amount of irregular end groups such as a succinimide end group in tPAA.16 In our previous study,26 we demonstrated that PAA hydrolase-1 selectively recognized amide bonds between β-aspartic acid units and hydrolyzed tPAA to yield oligo(aspartic acid) (OAA). In this study, we used three samples of R-poly(L-aspartic acid), tPAA, and the resultant OAA for hydrolysis by PAA hydrolase-2. When R-poly(Laspartic acid) was incubated for 24 h with PAA hydrolase2, the molecular weight of R-poly(L-aspartic acid) was clearly reduced, and concomitantly aspartic acid was generated, indicative of enzymatic hydrolysis of polymer (Figure 2a). However, when the tPAA was incubated with PAA hydrolase-2, the elution profile of treated tPAA was almost the same as that of original tPAA (Figure 2b). These results


Figure 2. Changes in molecular weights of R-poly(L-aspartic acid) (a), R,β-poly(D,L-aspartic acid) (tPAA) (b), and oligo(aspartic acid) (OAA) (c) treated by PAA hydrolase-2. R-Poly(L-aspartic acid) was incubated with PAA hydrolase-2 in 10 mM sodium borate buffer (pH 8.0) at 40 °C for 24 h, and tPAA and OAA were incubated with PAA hydrolase-2 in 10 mM sodium borate buffer (pH 8.0) at 30 °C for 48 h, respectively.

suggest that PAA hydrolase-2 had limited activity toward the tPAA chains via exo-mode because of the irregular end groups in the thermal synthesized tPAA.20 When the OAA, the hydrolyzed product of the tPAA via endo-mode by PAA hydrolase-1, was incubated with PAA hydrolase-2, it was hydrolyzed to yield aspartic acid (Figure 2c), suggesting that the freshly generated end groups of OAA could be hydrolyzed via exo-mode by PAA hydrolase-2. After further incubation of the reaction mixture with PAA hydrolase-2, however, a small amount of oligo(aspartic acid) remained as a final product together with aspartic acid. This result suggests that an additional PAA-hydrolyzing enzyme (PAA hydrolase-3) may participate in tPAA degradation in the cell of Sphingomonas sp. KT-1. Enzymatic Hydrolysis of r-Penta(L-aspartic acid) and β-Penta(L-aspartic acid) with PAA Hydrolase-2. To examine the substrate recognition of PAA hydrolase-2, the enzymatic hydrolysis of R- and β-penta(L-aspartic acid) with PAA hydrolase-2 was carried out, and the hydrolytic products were analyzed by HPLC. Figure 3a shows a typical HPLC curve of the hydrolytic products of R-penta(L-aspartic acid) with PAA hydrolase-2 in the reaction time of 30 min. The component of each peak was R-penta(L-aspartic acid) (5mer), R-tetra(L-aspartic acid) (4mer), or R-tri(L-aspartic acid) (3mer), whereas the peaks of R-di(L-aspartic acid) (2mer) and L-aspartic acid (1mer) were not separated from each other under the measurement conditions. Figure 3b shows the time dependence on the amount of products generated during the hydrolysis of R-penta(L-aspartic acid) with PAA hydrolase2. The amount of R-penta(L-aspartic acid) decreased with


reaction time, and the amount of R-tetra(L-aspartic acid) concomitantly increased. On the other hand, the peak of R-tri(L-aspartic acid) was hardly observed until the reaction time of 60 min. This result indicates that R-penta(L-aspartic acid) is hydrolyzed via exo-mode by PAA hydrolase-2. To elucidate the influence of amide linkage on oligo(aspartic acid) hydrolysis by PAA hydrolase-2, the hydrolysis of β-penta(L-aspartic acid) by PAA hydrolase-2 was carried out. A typical HPLC curve of the hydrolytic products of β-penta(L-aspartic acid) with PAA hydrolase-2 in the reaction time of 90 min is shown in Figure 3c. Under the measurement conditions, L-aspartic acid, β-di(L-aspartic acid), β-tri(L-aspartic acid), β-tetra(L-aspartic acid), and β-penta(Laspartic acid) could be separated from each other in contrast to the hydrolysis products of R-penta(L-aspartic acid). Figure 3d shows the time dependence of the amount of products generated during the hydrolysis of β-penta(L-aspartic acid) with PAA hydrolase-2. When β-penta(L-aspartic acid) was used as a substrate, the amount of β-penta(L-aspartic acid) decreased with reaction time, and the amount of β-tetra(Laspartic acid) concomitantly increased in the same way of β-penta(L-aspartic acid) as a substrate. This result also indicates that β-penta(L-aspartic acid) is hydrolyzed via exomode by PAA hydrolase-2. The rates of enzymatic hydrolysis of R-penta(L-aspartic acid) and β-penta(L-aspartic acid) were determined as 14.5 and 16.5 µM/min, respectively. Thus, the rate of hydrolysis of R-penta(L-aspartic acid) was similar to that of β-penta(L-aspartic acid). This result suggests that the hydrolysis of R- and β-penta(L-aspartic acid) by PAA hydrolase-2 is independent of the amide linkage in the oligomers. It is also speculated that PAA hydrolase-2 may recognize at least the C-terminal end group of the oligomers and hydrolyze them via exo-mode to generate aspartic acid because only the C-terminal end group of the oligomers are the same for Rand β-penta(L-aspartic acid). On the basis of the results of the present study and our previous work,26 we propose the degradation mechanism of tPAA in the cell of Sphingomonas sp. KT-1 as shown in Figure 4. First, PAA hydrolase-1 hydrolyzes specific amide bonds between β-aspartic acid units in tPAA via endo-type hydrolysis to yield oligo(aspartic acid) (OAA). The resultant OAA still contained both R- and β-amide bonds in the molecule because D- or L-monomeric units in tPAA may influence the hydrolysis by PAA hydrolase-1.26,27 Second, PAA hydrolase-2 attacks the resultant OAA and generates aspartic acid via exo-type hydrolysis. Taking the chemical structure of R- or β-penta(L-aspartic acid) and the newly generated end groups in the OAA into consideration, PAA hydrolase-2 may recognize the C-terminal end group of the oligomer and hydrolyze them via exo-mode to generate aspartic acid (Figure 4). Gene Analysis of PAA Hydrolase-2 from Sphingomonas sp. KT-1. The PAA hydrolase-2 gene from Sphingomonas sp. KT-1 was cloned as described in the Materials and Methods section. First, the DNA fragment corresponding to the internal region of the determined N-terminal amino acids of the mature PAA hydrolase-2 was amplified by a degenerated PCR. The resultant PCR product was inserted into

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Figure 3. (a) Typical HPLC curve of the products during the enzymatic hydrolysis of R-penta(L-aspartic acid) with PAA hydrolase-2 in the reaction time of 30 min. 1mer, L-aspartic acid; 2mer, R-di(L-aspartic acid); 3mer, R-tri(L-aspartic acid); 4mer, R-tetra(L-aspartic acid); 5mer, R-penta(L-aspartic acid). (b) Time dependence of the products generated during the hydrolysis of 1.0 mM R-penta(L-aspartic acid) with PAA hydrolase-2 at 30 °C. R-Penta(L-aspartic acid) (9), R-tetra(L-aspartic acid) (b), R-tri(L-aspartic acid) (2), and R-di(L-aspartic acid) and L-aspartic acid ([) were analyzed by HPLC at 210 nm. R-Di(L-aspartic acid) and L-aspartic acid were not separated from each other under the measurement conditions. (c) Typical HPLC curve of the products during the enzymatic hydrolysis of β-penta(L-aspartic acid) with PAA hydrolase-2 in the reaction time of 90 min. 1mer, L-aspartic acid; 2mer, β-di(L-aspartic acid); 3mer, β-tri(L-aspartic acid); 4mer, β-tetra(L-aspartic acid); 5mer, β-penta(L-aspartic acid). (d) Time dependence of the products generated during the hydrolysis of 1.0 mM β-penta(L-aspartic acid) with PAA hydrolase-2 at 30 °C. β-Penta(L-aspartic acid) (0), β-tetra(L-aspartic acid) (O), β-tri(L-aspartic acid) (4), β-di(L-aspartic acid) (]), and L-aspartic acid (3) were analyzed by HPLC at 210 nm.

Figure 4. Proposed mechanism for the hydrolysis of tPAA by two enzymes of PAA hydrolase-1 and PAA hydrolase-2 in the cell of Sphingomonas sp. KT-1.

pGEM-T Easy, and its sequence was determined. To obtain the whole PAA hydrolase-2 gene, LA PCR in vitro cloning was performed. For LA PCR in vitro cloning, four primers were designed from the amplified internal region that encodes the amino acid sequence LKTKGYEAAVKILDRDH. The fragments after LA PCR were cloned into pGEM-T Easy, and the nucleotide sequences 5′ and 3′ to the nucleotides

that encode the internal amino acids (LKTKGYEAAVKILDRDH) were determined. Unfortunately, no termination codon could be found at the position expected based on the molecular weight of the mature PAA hydrolase-2 protein. Therefore, genomic Southern hybridization and colony hybridization using DIG-labeled PCR product as a probe were carried out to clone the complete gene of PAA hydrolase-2. Plasmid DNA including ClaI fragment were positively hybridized to DIG-labeled PCR product and were screened for the PAA hydrolase-2 gene by Southern hybridization, and nucleotide sequencing was carried out to confirm that we had cloned the entire PAA hydrolase-2 gene sequence. Sequence analysis revealed that the PAA hydrolase-2 gene was composed of 1275 bp, and the open reading frame starts from the putative initiation codon ATG at nucleotide 1, located 6 bp downstream of the putative Shine-Dalgarno (S/ D) sequence. The predicted polypeptide encodes a preprotein of 425 amino acids with a predicted molecular mass of 44 647 Da. However, the first 21 amino acids likely encode signal peptide because the N-terminal sequencing of the purified protein revealed that the N-terminus matches that of the predicted amino acid residue at predicted position 21. Once this fact was taken into consideration, the molecular mass deduced from PAA hydrolase-2 gene was 42 584 Da which was in agreement with the value (42 kDa) determined by SDS-PAGE of the purified protein.



Figure 5. Alignment of the putative amino acids of PAA hydrolase-2 from Sphingomonas sp. KT-1 with those of putative peptidase from C. crescentus CB15 and carboxypeptidase G2 from Pseudomonas sp. strain RS-16. Putative active residues of PAA hydrolase-2 conserved among the three enzymes were marked by closed circles.

The deduced amino acid sequence of PAA hydrolase-2 was used to search the protein database by the FASTA program. The result of FASTA showed that the amino acid sequence of PAA hydrolase-2 was highly similar with that of a putative peptidase belonging to the M20/M25/M40 family of metallopeptidases from C. crescentus CB15 (63.4% identity in 413 aa) (Figure 5).34 This finding suggests that PAA hydrolase-2 may contain metal ion(s) within the active site and corresponds to the fact that the hydrolysis of R-di(L-aspartic acid) with PAA hydrolase-2 was inhibited by EDTA. However, no significant homology of the deduced amino acid sequence of PAA hydrolase-2 was detected with PAA hydrolase-1, intracellular and extracellular cyanophycin hydrolases that degrade multi-L-arginyL-poly(L-aspartic acid) (cyanophycin).35 The deduced amino acid sequence of PAA hydrolase-2 had similarity with that of carboxypeptidase G2, which hydrolyzes the C-terminal glutamate moiety from folic acid and is a zinc-dependent homodimeric exopeptidase, from Pseudomonas sp. strain RS-16 (27.5% identity in 435 aa).36,37 The crystal structure of the carboxypeptidase G2 has been determined.38 This crystal structure shows that in the active site of the carboxypeptidase G2 two zinc ions (zinc 1 and zinc 2) were coordinated by one histidine, one glutamate, and one aspartate ligand, respectively. One zinc ion was coordinated by the carboxylate oxygens of Asp141and Glu176 and by His385, whereas the other was coordinated by the other carboxylate oxygen of Asp141, by carboxylate oxygen of Glu200 and by His112. When the amino acid sequences of PAA hydrolase-2, peptidase from C. crescentus and carboxypeptidase G2 from Pseudomonas sp. were aligned, several amino acid residues (His114, Asp141, Asp142, Glu175, Glu176, and Arg336 in PAA hydrolase-2) were highly conserved among the three

sequences (Figure 5). Based on the crystal structure of carboxypeptidase G2, the alignment suggests that His114, Asp141, and Glu176 of PAA hydrolase-2 may act as metal ligands. It has been proposed that Glu175 in carboxypeptidase G2 plays the role of a general base in hydrolytic catalysis; therefore, Glu175 of PAA hydrolase-2 may also act as a general base in the reaction mechanism. Moreover, Rowsell et al. have demonstrated that Glu175 and Glu176 in carboxypeptidase G2 forms a cis-peptide bond which is conserved among exopeptidases, so that Glu175 and Glu176 in PAA hydrolase-2 may also form a cis-peptide bond.38 The charged residue Arg324 of carboxypeptidase G2 is speculated to participate in binding to the glutamate side chain of the substrate in their previous work. Therefore, it is suggested that Arg336 of PAA hydrolase-2 may be involved in substrate binding. In this way, the sequence alignment has shown that the several amino acid residues, which participate in the catalytic reaction of carboxypeptidase G2, are strictly conserved in the deduced amino acid sequence of PAA hydrolase-2, suggesting that these enzymes originated from an ancestral peptidase and divergently evolved from a common ancestor. Conclusions This paper has reported the purification, characterization, and genetic analysis of poly(aspartic acid) (PAA) hydrolase-2 from Sphingomonas sp. KT-1 (JCM10459). The effect of inhibitors showed that the R-di(L-aspartic acid) hydrolysis activity of PAA hydrolase-2 was significantly inhibited by the metalloprotease inhibitor EDTA. GPC analysis showed that R-poly(L-aspartic acid) was hydrolyzed to generate aspartic acid by PAA hydrolase-2, whereas only a small

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amount of tPAA was hydrolyzed by PAA hydrolase-2, suggesting that the irregular end groups in tPAA may inhibit the activity of the enzyme. Reversed-phase HPLC analysis of the products of hydrolysis of R- and β-penta(L-aspartic acid) by PAA hydrolase-2 demonstrated that the exo-mode of hydrolysis of R- and β-penta(L-aspartic acid) occurred. Previously isolated PAA hydrolase-1 hydrolyzed β-β amide bonds in the tPAA chain via endo-mode to yield oligo(aspartic acid) (OAA), and the newly generated end units of the OAA were hydrolyzed to aspartic acid by PAA hydrolase-2 via exo-mode. It is concluded that tPAA is hydrolyzed to OAA by PAA hydrolase-1, followed by the hydrolysis of the resultant OAA by PAA hydrolase-2 to aspartic acid as shown in Figure 4. The results on the tPAA hydrolysis by PAA hydrolase-1 and PAA hydrolase-2 support the structural effects on biodegradability of tPAA in active sludge, in which the amount of irregular end groups in tPAA influences on the biodegradability of tPAA. Moreover, many sphingomonads have been isolated that are capable of degrading xenobiotic and may play important roles in the microbial degradation of man-made xenobiotics liberated in the environment.39 Therefore, these findings may help us to design new types of PAA to control biodegradability of the polymer. Genetic analysis has demonstrated that the deduced amino acid sequence of PAA hydrolase-2 is similar to that of the putative peptidase from C. crescentus CB15 and carboxypeptidase G2 from Pseudomonas sp. strain RS-16. An alignment of these three proteins has indicated that PAA hydrolase-2 has several conserved amino acid residues corresponding to the amino acid residues which play important roles in the catalytic reaction of carboxypeptidase G2. Therefore, these residues may also play a role in the catalytic function of PAA hydrolase-2. Acknowledgment. We gratefully acknowledge Dr. B. Mohr of BASF, Germany for supplying the PAA sample, and appreciate the assistance provided by Dr. C. Nomura for the English correction of our manuscript. This research was supported by a grant for Ecomolecular Science Research to RIKEN Institute and a SORST (Solution Oriented Research for Science and Technology) grant from the Japan Science and Technology Corporation (JST). References and Notes (1) Loiseau, J.; Doeerr, N.; Suau, J. M.; Egraz, J. B.; Llauro, M. F.; Ladaviere, C.; Claverie, J. Macromolecules 2003, 36, 3066. (2) Buchholz, F. L. Polyacrylamides and poly(acrylic acids). In Industrial Polymers Handbook; Wilks, E. S., Ed.; WILEY-VCH Verlag GmbH: Weinheim, Germany, 2001; p 565. (3) Oppermann-Sanio, F. B.; Steinbu¨chel, A. Naturwissenschaften 2002, 89, 11. (4) Swift, G. Polym. Degrad. Stab. 1998, 59, 19. (5) Kunioka, M.; Choi, H. J. Polym. Degrad. Stab. 1998, 59, 33. (6) Oppermann, F. B.; Pickarts, S.; Steinbu¨chel, A. Polym. Degrad. Stab. 1998, 59, 337. (7) Gross, R. A. Bacterial Poly(γ-glutamic acid). In Biopolymers from Renewable Resources; Kaplan, D. L., Ed.; Springer: Berlin, 1998; p 195.

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BM034085I

Biochemical and Molecular Characterization of Poly (aspartic acid

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