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Cloning and sequence analysis of a polyurethane esterase of Comamonas acidovorans TB-35
JOURNALOF FERMENTATION ANI) BIOENOINEERING Vol. 86, No. 4, 339-345. 1998
Cloning and Sequence Analysis of a Polyurethane Comamonas acidovorans TB-35 NOBUHIKO
NOMURA,
YUKIE SHIGENO-AKUTSU, TOSHIAKI AND TADAATSU NAKAHARA
Institute of Applied Biochemistry,
Esterase of
NAKAJIMA-KAMBE,*
University of Tsukuba, Tsukuba, Ibaraki 305, Japan
Received 13 June 1998/Accepted 6 July 1998 Comamonas acidovorans strain TB-35 has an esterase that degrades solid polyester polyurethane (PUR). The structural gene,pudA, for the PUR esterase has now been cloned in Escherichia coli. WhenpudA was expressed in E. coli, the recombinant protein was able to degrade solid PUR. The predicted amino acid sequence contains the Gly-X1-Ser-X2-Gly motif characteristic of serine hydrolases. The highest degree of homology was detected with the Torpedo californica acetylcholinesterase (T AChE), possessing the Ser-His-Glu catalytic triad, with the glutamate residue replacing the usual aspartate residue. Similarity in the number and positions of cysteine and salt bonds was very apparent between PudA and T AChE, as were also identities of sequences and their positions in the a-helix and &strand regions between the two. In the neighborhood of the glutamate residue of the Ser1~-His@3-Glu3” catalytic domain of PudA, there were three hydrophobic domains, one of which constituted the surface-binding domain, which occurred in the C-terminus of most bacterial poly(hydroxyalkanoate)(PI-IA) depolymerases. [Key words:
Comamonas acidovorans, polyurethane, esterase, cloning, nucleotide sequence]
Polyurethane (PUR), which is a kind of plastic, is widely used as a base material in various industries. PUR is an artificial synthetic polymer which is waterinsoluble (hydrophobic), and is generally synthesized by condensation of polyol and polyisocyanate. An estertype PUR degradation activity has been reported in several microorganisms, but the enzymes have not yet been purified, or characterized in detail (l-4). In our previous papers, we reported the isolation of a bacterium, Comamonas acidovorans TB-35, which could utilize solid polyester PUR as the sole carbon and nitrogen source (5). We also found that an esterase played a major role in PUR degradation by this strain (6). This strain had two kinds of extracellular esterases; one secreted into the culture medium and the other, bound to the cell surface. Only the cell-bound esterase could degrade PUR. Recently, we succeeded in purifying the cell-bound esterase (PUR esterase) and reported that this enzyme probably degrades PUR in a two-step reaction; hydrophobic adsorption onto the solid PUR surface and hydrolysis of the ester bonds of PUR (7). Regarding solid polyester biodegradation, it is well known that poly(3-hydroxyalkanoate), PHA, can be degraded by a large variety of microorganisms (8-15). The key enzyme for PHA degradation, PHA depolymerase, is also a kind of esterase. Almost all PHA depolymerases consist of two separate domains, the catalytic domain and the PHA surface-binding domain (16-18). It has been reported that the structure of the PHA depolymerase is such that inhibition of the degradation activity occurs in the presence of excessive amounts of the enzyme (19). However, we recently demonstrated that the degradation activity of the PUR esterase (PudA) did not decrease but rather remained constant when an excess of the enzyme was present (7). It was also indicated by enzymatic characterization that the PUR esterase was a novel plastic-degrading esterase
with properties different from those of lipases or PHA depolymerases. In this study, we present the molecular cloning and sequence analysis of the PUR esterase gene in C. acidovorans TB-35, and its functional expression in E. coli. By sequence comparison, it was shown that the amino acid sequence of the PUR esterase was similar to that of the Torpedo californica acetylcholinesterase (T AChE), and very different from that of the PHA depolymerases except in respect of the PHA surface-binding domain. The putative structure and characteristics of the PUR esterase encoded by the gene are discussed. MATERIALS
AND METHODS
Materials The polyester polyurethane (PUR) used in this study was synthesized by the condensation of poly(diethylene glycol adipate) (DEGA) and 2,4-tolylene diisocyanate under anhydrous conditions as described previously (6, 7). The DEGA was supplied by Suzuki Motor Co. (Hamamatsu). E. cofi DHlOB was purchased from Toyobo Co. Ltd. (Osaka). All other compounds used were standard commercial preparations. Bacterial strains, plasmids, and media The culture conditions for C. acidovorans TB-35 have been described previously (5). E. coli DHlOB and JMl09 were used for the isolation and maintenance, respectively, of recombinant derivatives of pUC19 (20). E. coli strains were grown at 37°C on Luria-Bertani (LB) medium (21) with appropriate supplements. Ampicillin (final concentration of 100 pg/ml) was added to the medium, when necessary, for plasmid selection. The expression of the PUR esterase gene in recombinant E. coli strains was tested on an indicator plate. The plate was prepared by pouring an overlay of 1.5% agar solution containing a sonicated emulsion of 0.5% DEGA onto a previously prepared LB agar plate (7). Enzyme activity assays The esterase activity for a water-soluble substrate (p-nitrophenyl acetate) was deter-
* Corresponding author. 339
340
NOMURA
J. FERMENT. BIOENG.,
ET AL.
mined by the method of Kay et al. (22). One unit was defined as the amount of enzyme required to liberate 1 /*mol of p-nitrophenol per min. The PUR degradation activity was determined by the procedure described previously (6, 7). Samples (0.5 ml each) of 1OOmM potassium phosphate buffer (pH 7.0) which contained cell-free extracts of recombinant E. coli culture media were transferred to small test tubes. The reaction was started by adding a PUR block (4 x 4 x 1 mm) to each of the tubes. After 48 h incubation at 3O”C, the PUR blocks were removed, and the amount of free diethylene glycol produced by hydrolysis of the ester bond, was quantified by gas chromatography (GC) using a model GC-8A gas chromatograph (Shimadzu Co., Tokyo), as described previously (6). Preparation of the cell-free extract from recombinant One loopful of cells of recombinant E. coli E. coli JM109 was inoculated into a 5OOml shaking flask containing 1OOml of LB medium (100 /*g/ml ampicillin). After incubation for 5.5 h at 37”C, cells were harvested by centrifugation at 8000 x g for 10 min. The cells were suspended in 20 mM potassium phosphate buffer (pH 7.0) and disrupted using a sonicator. The disrupted suspension was centrifuged at 12,000 x g for 20 min to remove cell debris and unbroken cells. Protein SDS-PAGE analysis was Protein analysis performed as described by Laemmli (23). Proteins were electroblotted from SDS-polyacrylamide gels onto a polyvinylidene difluoride membrane (PVDF) (Clear Blot Membrane-P, ATT0 Co. Ltd., Tokyo) and the N-terminal sequence was determined using an automated protein sequencer (model 492, Parkin Elmer Applied Biosystems, Tokyo). General molecular procedures and computer analysis Unless specified otherwise, standard methods for DNA manipulation described by Maniatis et al. (21) were followed. Southern hybridization was carried out using hybridization kits (ECL 3’-Oligolabelling and detection system, Amersham, London, UK). An ABI PRISM Dye Terminator Cycle sequencing Core kit (Parkin Elmer Applied Biosystems) was used for DNA sequencing. The products of the termination reactions were analyzed with the aid of an automated DNA sequencer (model 373A, Parkin Elmer Applied Biosystems). Sequence comparisons were made using the EMBL and GenBankTM databases. The nucleotide and amino acid sequences were analyzed using the GENETYX-MAC program, version 8 (Software Development Co. Ltd., Tokyo), FASTA (ver. 3.0), and BLAST (ver. 2.0). The predicted secondary structure of PudA was calculated by the DDBJ SSTHREAD program. The nucleotide sequence data reported in this paper can be obtained from the DDBJ nucleotide sequence database with the accession number AB009606.
entire genome of C. acidovorans TB-35, completely was hybridized with the digested with EcoRI, oligonucleotide probe on a Southern blot, to produce hybridized fragments of about 10.0 kbp (data not shown). Fragments with a size of about 10.0 kbp were recovered from the agarose gel and ligated with the EcoRI-digested and dephosphorylated vector, pUC19. The ligation mixture was used to transform E. coli DHlOB. About 50 recombinant clones were screened by Southern hybridization of the EcoRI-digested plasmid DNA using the oligonucleotide as a probe. A 10.0 kbp EcoRI-digested plasmid DNA fragment from each clone specifically hybridized with the probe and indicated that the 5’ region of the PUR esterase had been cloned. The clones expressed esterase activity in E. coli and produced a translucent clear zone on an indicator plate containing DEGA. To define the location of the gene on the 10.0 kbp fragment that hybridized with the probe, the fragment was digested with restriction enzymes. The fragments were subcloned into the appropriate sites of the pUC19 vector. The resultant plasmids were tested for their abilities to produce clear zones on an indicator plate. The gene responsibIe for clear zone formation was located in pUR22, which had the smallest length of DNA, of less than about 2.2 kbp. Functional expression of p&A gene in E. coli To investigate the biochemical properties of the recombinant PUR esterase, the protein was expressed in an E. coli strain carrying pUR22. The recombinant protein separated by SDS-PAGE is shown in Fig. 1. It was observed that the apparent molecular masses of both the recombinant PUR esterase and the native PUR esterase purified from C. acidovorans TB-35 were identical. No relevant band was observed at the same mobility in the lane containing the extract from E. coli carrying pUC19. The recombinant PUR esterase was tested for its ability to degrade PUR. The esterase activity of the cell-free extract of the recombinant E. coli carrying pUR22 was 0.3 U/mg protein. The PUR cube was degraded by the extract producing diethylene glycol (50 pmol/mg protein/48 h), a degradation product which was detected by GC analysis. The cell-free extract of E. coli carrying pUC19 did not possess the esterase activity and degrada-
kDa
1
2
3
4
RESULTS Cloning of the gene encoding the PUR esterase Previously, we purified the cell-bound esterase, a PUR degradation enzyme, from C. acidovorans TB-35 (7). The amino acid residues at the N-terminus of the PUR esterase were sequenced (N-(C)GGSDNDSSSNNQGAPA VA-C). An oligonucleotide (5’AAC GA(G/C) AGC TCG AGC AAC AAC CAG GGC-3’) was synthesized using 9 amino acids (NDSSSNNQG) present in the N-terminal 19 amino acids of the PUR esterase taking into consideration Comamonas species codon preferences (18). The
20.1 14.4 FIG. 1. SDS-PAGE analysis of the crude extract from E. co/i containing pUC19 or pUR22, respectively (lanes 2 and 3), and of the purified esterase from C. acidovorans TB-35 (lane 4). The sizes in kilodaltons of the molecular weight markers (lane 1).
CLONING
VOL. 86, 1998
OF COMAMONAS
POLYURETHANE
-339
GTCGCAAGGCGAGCCGGATTGGGATCTGGCAGCTCT~CAGCTC~CCCGACGA~TAGACCAGCGCGTC~TT~TGACCCAGTGA
-261
AGCGGCGGTATCCATCGCTGCGGGGAAGCAGCTGCGCGCGCGCGCGCCAGGCCC~TGCATACT~CTCCC~GCTCTTGC
ESTERASE
GENE
341
-262 -184
-35 -183
CATT(;AGCGGCAAGCGAGCGCTCAACCTTCCTTCCGACCT
-106
-10 -105
-27
+PUa ATACl3AAGATCCTTC~ATTG~TGAATGAATAGCCGTTCCCT~CT~GCGATCCGATTTCCCAC~TTCTTGCA MNSRSLTKAI
S.D.
52
F
129
SSNNQGAPA 207
RLGMRRYFG
TVAGQVQAVD
I
P
F
A 285
GCGCCTCCGGTGGGCAACCTGCGCTGGATGCCGCCGCCGGCGCCGCCTC~TCCTGGGCCGCCCCGCTGGCG~GACCCAG 4
286
SVLGACGGSDNDS
GTTGCCATTACGGTCGCAGGCCAGGTCCAGGCTGTGTGGACCGCCTCGGCATGCGCAGATATTTCGGGATACCTTTTGCC VAI
208
51 RFPTILA
CTCGCCGGCTTCAGCGTACTTGGTGCATGCGGTGGCAGCGAC~CGACAGCAGTAGT~C~CCAG~CGCACCCGCC LAG
130
-28
CATGCGGTTGAATTTCCTATCCGTGGTAGCATCGTTCGCATCGTTCGCGCGG~T~T-C~CATTTCGCA~GT-CAC
P
PVGNLRWMPPAPPQSWAA
PLAKTQ 363
TCTAATGCGCCCTGCATGCACCGGCGCCACCGATCCCCTCCGCCTGCCC~CGGCACCGA~ACTGCCTCTACCTC SNAPCMQTGATDPLRLPNGTEDCLYL
364
441
~ACGTCCATGCCCCTGCCACCGGCGAAGGCCCCCTTCCCTGTCATGGTGTGGATCCACGGC~CGCCTT~AGCATCGGC DVHAPATGEGPFPVMVWIHGGAFSIG
442
G 520
519
GGCACCATCACCTATGCCGACCCGTCGCCCCTGGTCAGCAGGGC T
PLVSKGVIVVN
ITYADPS
IAYRMG 597
GCCATGGGTTTCCTGGGCCATCCGTCGCTGCGCGCCGCGCACCACGGTC~C~CTACGGCATCAT~ACCAGCAG AKGFLGHPSLRAADGTVGNYGIMDQQ
598
A 676
WV
Q
D
N
IAAFGGDKSNVTI
F
C:
G
F
753
ci
YGFDRQ
LTQAQL
E
A
Q
S
A
A
G
V
S
C
AT
A
F
T
TAN
W
S
P
V
P
S
V
D
G
V
L
P
K
987 S
I
K 1065
DEWSYFV
:j
L
RELVAGPLTAAQYPSYLQTS
1143 G
L
s
8:
Y
1221
1299 SQATPIF
TDMHFSCPALNLSKRVL
E
L
1377
SFNQGAGH
IGRNTI
Q
YLFNLRDLETAEHRDLQA
1455 S
MA 1533
CGCTACTGGACCAACTTTGCCCGCACCAGCAATCCGAATATTACG SWPAFT
YWTNFARTSNPNNGDPVAT
1611
GGACCGACCAAGGTGCTCGGACTGGACGTCGCCTCCGCTGGCGGCATCAG~~CTGGCCACGTTCGAGACCGACCAC IRELATFETDH
PTKVLGLDVASAGG
1689
AAG'TGCAACACGGCCTGGACATCACTGACTTTTTGATCACCTTTTT K
':
NT
A
P
SLAATA
LATVYPLTDYGTNTAQQP
ACGTATGAGCTCCAGTACCTGTTCAACCTGCGTGACCTGCGTGACCTGGAGACCGC~~CACCGCGACCTGCAGGCCAGCATGGCA
G 1612
K
ATGTACGAGTTCCGGGACCGGACTGCGATCCCGTCGATCGGCCGC~CACTATCAGTTTC~CCAG~GGCAGGTCAC
R 1534
909
GCC';GCACCGACATGCACTTTTCCTGTCCGGCGTTGAACTCTTC
T 1456
831
IVNSAL
PTVDAACLRGLSAELVNNQ
MYEFRDRTAIPS 1378
S
CCA'rCGCTCGCCACGGTATATCCACTGACGGATTACGGCACC~TACCGCGCAGC~CCCAGCCTC~GGCGACGGCG
A 1300
T
GCATCGCGCGAACTGGTGGCGGGGCCGCTGACCGCTGACCGCTGCCCAGTACCCTTCCTATCTGCAGACCTCGCTCGGTCTGCCA
P 1222
S
GCGACTTTCGTCGCCGGGGAGAACAACAAGGTGCCGCTGGTTCGTT
A 1144
E
PLSKGLFAKAIVQS
SVMTHLAS
ATFVAGENNKVPLVNGSNQ 1066
G
CTCGCGACCGCATTCACCACGGCCRACTGGAGCCCCGTGGAGCC~CGTGCCTTCGGT~ATGGC~GGTTTTGCCC~GTCCATC~G L
988
R
GCCOCAGCCGGCGTTAGCTGCCCCACCGTCGACGCGGCATGTTTGCGCGGCTTGTCCGCCGAGCTTGTC~C~CCAG A
910
L
GGCC:GCTACGGATTCGATCGCCAGCTGACACAGGCGCAGCTTG~GCGCAGAGCACGTCGATCGTC~CAGCGCGCTG G
832
A
GCCGGCGGCTTCAGCGTGATGACACACCTGGCGTCACCGCGC A
754
675
GCAGCACTGCGCTGGGTGCAGGACAATATCGCCGCGTTCGCCGCGTTCGGCGGCGAC~GTCC~TGTCACCAT~TTCGGCG~TCC
W
T
S
L
T
F
dt--
1690
TATTGCACCGCATGACGTTTGGGACGCAAGTCATTCATTCCTTC~GATACTCGCGCAGTC~CCCT~ACCATTTCCAGTT
1768
CCT~ACACACCAGATGAGACCGTTGGTTTTCTCTGGCAAGCATGC
1767 1835
FIG. 2. Nucleotide sequence of PUR &erase. The deduced amino acid sequence is shown under the nucleotide sequence. A putative promoter region, Shine-Dalgano sequences (S. D.), and p-independent terminator are indicated. The chemically determined N-terminal amino acid sequences of the PUR esterase purified from C. acidovorans TB-35 are indicated with a single underline.
342
NOMURA
ET AL.
J. FERMENT. BIOENC.,
tion activity of PUR. These results indicate that the gene encoding for the PUR esterase is included in the DNA fragment inserted in pUR22. Nucleotide sequence and deduced product of the DNA that encodes the PUR esterase The nucleotide sequence of the 2174 bp DNA fragment inserted in pUR22 was determined for both strands. The complete nucleoS
tide sequence of the sense strand and the deduced amino acid sequence are shown in Fig. 2. Computer analysis of the nucleotide sequence detected one open reading frame (ORF), located between nucleotides 1 and 1644. The molar G+C content was 63.4%. At the third position of the codon, G or C was found in 78.9% of the cases. The ORF consists of 1644 bp with a putative ATG initiation h-
S-
s
MPPAPPQSWAAPL-AKTQSNAP ** l
PUdA
1:CGGSDNDSSSNNQGAPAVAITVAGQVQAVDRLGMRRYFGIPFAAPPVGN ***** * * *
T AChE
l:-----DDHSELLVNTKSGKV-MGTRVPVLSG-HISAFLGIPF~PPVGN~R~EPKKPWSG~AST~N-~QYVD
s-s
-9
*****
w
72
s-
3
L
s
b
9,
PUdA
77:---TG-ATDPLRLPN-G*
YLDVHAPATGEGPFPVHVWIHGGAFSIG-GTI-TYADPSPLVSKGVIWNIA ***** ** * * * * *
T AChE
73:EQFPGFSGSEMWNPNREM
YLNIWVPSPRPKSTTVMVWIYGGGFYSGSSTLDVYNGKYLAYTEEWLVSLSY s-s
e-h
s-
h-s-
PudA T AChB
153:FGFL---ALHGS
I -h
II
h-h
PUdA
2 2 7 : GYGFDRQLTQAQLEAQS
T AChE
228:SPNCPWA-----SVSVAEGRRRAVELGRNL
--
2 99 KKPQELIDVEW’NVLPFDSIFRFSFVPVIDGEFFP
h
M-h
h
302
h-h
III h PUdA T AChE
h
+
s-s
h-h
300:KSIKATFVAGENNKVPLVNGSNQDEWSYF-V-ASRE l * * * * l * * *
**
h-h
s-s
h
*
h-
h-
T AChE
h *
**
-GTN *
303:TSLESMLNSGNFKKTQILLG~KD~GSFFLLYG~GFSKDSRSKISR~DFMSG~LS~HANDLGLDA~LQYTD~DDN -h
PUdA
h
h
382
h-
+
S---8
371:TA-QQP-SLAATAAGTDMHFS *
~NLSKRVLSQATPIFMYEFRDRTAIPSIGRNTISFNQGAGHTYELQYLFN--L-RD * ** ** ***
383:NGIKNRDGLDDI-VG-DHNVI
LMHFVNKYTKFGNGTYLYFFHR-A-SNLVW-P-EW-MDV~ h
h
445 *
l
455
s-
-h
h-
S----8
522
PudA * T AChE
370
***
*
**
**
*
***
**
**
*
*
456:LNYTAgEE~SRRIMHYWATF~TGNPNEPHSQESK~LFTTKEQKFIDLNTEPMKVHQRL----RVQ h S” sh
FWNQFLPKL h
531
h
PudA T AChE
532:LNATACDGELSSSGTSSSKGIIFYVLFSILYLIF
FIG. 3. Comparison of the (26). Residues that are identical Regions 1, II, and III are shown under the sequence, respectively. Cys-Cys bridges and conserved
amino acid sequences of C. acidovoruns PUR esterase (PudA) and T. californicu acetylcholinesterase (T AChE) in both sequences are indicated by an asterisk (single-letter amino-acid notation). Gaps are indicated by hyphens. in black boxes. The secondary structure elements observed in PUR esterase and T AChE are shown above and a helix is shown as h-h; ,9 strand is shown as s-s; + indicates residues from the catalytic triad; the pattern of salt bridges are also shown by boxes.
CLONING OF COMAMONAS
VOL. 86, 1998 codon
that is 7 bp downstream from a potential ribosome-binding site (~ 12AGGAGA-7). No other putative start codons were found in the nucleotides upstream. Examination of the nucleotide sequences for the presence of a p-independent terminator of transcription revealed a stem and a loop followed by a short stretch of thymidine residues (1667GGCCCGC----GCGGGCCTT TTTTATTL693). A potential promoter sequence similar to the -35 and ~ 10 consensus sequences of E. coli was found upstream of the ORF, although the transcription start site has not yet been mapped. The nucleotide sequence of the ORF coded exactly for the chemically determined N-terminal amino acid sequence ((C)GGSD* l) of the purified PUR esterase from C. acidovorans TB-35 (Fig. 2). The ORF for the PUR esterase contained a putative start codon, 26 amino acids upstream of the experimentally determined N-terminal codon. The ORF encodes 522 amino acid residues capable of forming a 55,110 Da processed protein. It was concluded that this ORF coded for the PUR esterase and was named pudA. Analysis of the deduced amino acid sequences of pudA The first 26 deduced amino acids showed the characteristics of signal peptides of secretory precursors; three positively charged amino acids, arginine at positions 4 and 11 and lysine at position 8, were followed by a core of mainly hydrophobic amino acids (Fig. 2). The mature PUR esterase from C. acidovorans TB-35 has the sequence (Cys’)-Glyz-Gly3-Se&Asps at the N-termini. Ala-’ is a well-known cleavage site for known leader peptidases (24). From computerized searches of databases, it was shown that PudA possessed a high degree of homology only with the catalytic regions of the serine hydrolase family proteins which contain the Ser-His-Glu catalytic triad with a glutamate residue replacing the usuai aspartate residue (25-28) (Table 1). Comparison of the positions of each residue of the Ser-His-Glu catalytic triad showed that the amino acid residues of PudA were in similar positions to those in the Torpedo californica acetylcholinesterase (T AChE) and the human cholinesterase (H ChE). In particular, PudA is 30.1% identical to T AChE (Fig. 3). Comparison of the positions of Cys residues in PudA and T AChE showed that the disulfide bonds were in identical positions (positions 74-92, 258-265, and 390-513). We carried out the calculation of the secondary structure of PudA by computer analyses and the result was compared with that of T AChE which was already determined by three-dimensional analysis (26). It was shown that the number and positions of the a-helix regions and j-strand regions in PudA were similar to those in T AChE (Fig. 3). Predictable identification of the PUR surface-binding domain We have already demonstrated, by enzymatic characterization, that the PUR esterase possesses highly l
POLYURETHANE
ESTERASE GENE
343
Region II I of PudA xi9
321 ~~QDEWSYFVASRELVAG-PLTAAQY~SYLQTSLGLPPSLATXJY~LTDPGT . ...** * *. . .*.* .*..*.* ***. . . ATvTNHYVAGRINVTQYNvLGARY-GYVl'TPLYYCPSLSGwTDKANCPI
PhaZi p/e PHAsurface-binding domain FIG. 4. Comparison with amino sequences of the PHA surfacebinding domain. Sequence alignment of C. acidovoruns PUR esterase (PudA) and the PHA surface-binding domain of the PHA depolymerase (PhaZlpk) (18). Identity and similarity of sequences are shown as * and , respectively. hydrophobic region(s) and that the hydrophobic adsorption of these region(s) onto the surface of the PUR is important for PUR degradation (7). Analysis of the primary structure of PudA revealed that PudA possessed three regions (region I, II, and III) containing many hydrophobic amino acid residues, in the center of its polypeptide chain (Fig. 3) and that the amino acid sequence of one (III) of the three regions was highly homologous to that of the PHA surface-binding domain of PHA depolymerase (PhaZlple) of Pseudomonas femoignei (Fig. 4) (18). No identity or similarity between PudA and PHA depolymerases was observed except in the PHA surface-binding domain. The three regions in this aforementioned domain include many hydrophobic amino acid residues different from those in T AChE which cannot degrade PUR. The hydropathy profiles for PudA and T AChE were similar except in the three regions (I, II, and III) (data not shown).
DISCUSSION We have cloned the structural gene (PudA) encoding the cell-bound PUR esterase from C. acidovorans TB35, and analyzed its sequence. It was demonstrated that recombinant PUR esterase could be expressed in E. coli as an active protein for the degradation of solid PUR. The amino acid sequence of pudA revealed no significant homology to sequences of PHA depolymerases except within the PHA surface-binding domain. PudA has regions similar to those of proteins in the family of serine hydrolases which possess the Ser-His-Glu catalytic triad (Table 1). Although alignments of PudA never showed identity of more than 30.1% (T AChE) in amino acid sequences, the sequence similarity between PudA and T AChE extends throughout the complete amino acid sequence of these proteins, being especially pronounced in some regions with a high degree of conservation. Also, the positions of Cys residues in PudA and T AChE were similar (Fig. 3). In T AChE, the sequence includes three pairs of Cys residues (position 67-
TABLE 1. Aligment of amino acid sequence of PUR esterase with the sequences of four Ser-His-Glu type serine hydrogenases Serine (S)” PudAb
T AChE HChE CCL1 PnbA Consensus
199 200
198 218 167
Glutamate (E)
FGGDKSNVTIFG~SAGGFSV FGGDPKTVTIFGESAGGASV FGGNPKSVTLFGESAGAASV FGGDPDKVMIFGESAGAMSV
324 327 325 354
GSNQDmSYF GVNKDEGSFF GVNKDEGTAF GNQEDEGTAF
FGGDPDNVTVFG~SAGGMSI FGGdp--Vt*FGl#SAG*-Sv
288
GTTRDZGYLF G---DEg*-F
Histidine (H) 433
440 438
GAGHTYELQYLF GVIBGYEIEFVF GVMHGYEIEFVF
463 377
GTFHGNELIFQF KAFEALELPFVF
g--H--E*_f-F
a The positions of serine, glutamate and histidine in the primary sequences of the corresponding mature proteins are indicated. b PUR esterase from C. acidovorons TB-35 (PudA), acetylcholinesterases of T. californicu (T AChE) (29), cholinesterase of humans (H ChE) (30), Geotrichum cundidum lipase isoform1 (GcLl) (31), p-nitrobenzyl esterase from Bacillus subtifis (PnbA) (32).
344
NOMURA ET AL.
J. FERMENT. BIOENO.,
-2
PudA
-21 1
200
T AChE
n
I
E 327
1
I
H440
1
.
I
-3
PhaZl me
PhaZppi
B
zie ;;;m$ne-rich
1
Lipase box
a
;;M;;;;;dule
l l
of
Cysteine
m
Disulfide bond
;;/;;rface-binding
II
FIG. 5. Comparison of the domain structures. C. ucidovorum PUR esterase (PudA), T. culifornicu acetylcholinesterase bacterial PHA depolymerases (PhaZple and PhaZppO (18, 35).
94, 254-265, and 402-521) that were found to participate in the stable formation of intra-molecular disulfide bonds (33). Between PudA and T AChE structures, the identities (or the similarities) and positions of sequences are also very apparent in the a-helix regions, p-strand regions, and salt bonds (Fig. 3). This suggests that the protein folding and the secondary structures of these enzymes are similar and therefore also share a common three-dimensional fold. Almost all PHA depolymerases consist of two separate domains, the catalytic domain and the PHA surface-binding domain (17, 34) (Fig. 5). These domains are linked by a flexible linker, which is a threonine-rich region (35) or a fibronectin type III-like module (Fn3), which is a eukaryotic extracellular matrix protein (36). By the previous biochemical characterization of the PUR esterase, we hypothesized that the surface-binding site and catalytic site of this enzyme existed nearby in three-dimensional space, unlike those of the PHA depolymerase (7). It was estimated here that PUR esterase included three putative surface-binding domains including many hydrophobic residues in the center of the enzyme polypeptide chain (Fig. 5). As an enzyme that possesses a structure that resembles PudA, the structure of T AChE differs from that of the PHA depolymerases in the catalytic and binding domains constructed by three-dimensional analysis (26). The flexible linker domain was also not observed in PudA and T AChE (Fig. 5). By these estimations and the remarkable structural similarities with T AChE, it is concluded that the structure of PudA is not similar to that of the PHA depolymerases and that the three putative binding domains of PudA are positioned close to the catalytic domain in three-dementional space. The conclusions support our hypothesis based on the enzymatic characterization of the PUR esterase. It would seem that PudA and the PHA depolymerases have evolved from distinct origins because of the differences in the structures of both enzymes. PUR esterase and PHA depolymerases are of interest primarily because of their structures facilitating the adsorption of insoluble solids. It was clearly demonstrated
(T AChE) (26), and
that a hydrophobic domain (surface-binding domain) of the C-terminus of a PHA depolymerase binds to solid PHA (37). We also demonstrated that the purified PUR esterase has hydrophobic region(s) and that the hydrophobicity is important for the adsorption onto PUR. In this study, it was shown that PudA possesses three domains which contain many hydrophobic residues and region III of the three hydrophobic regions of PudA is similar to the PHA surface-binding domain of the PHA depolymerase. It was concluded that all or some of the three regions could play a role in the binding of PUR. Although the structures of PudA and the PHA depolymerases are probably be very different, both enzymes possess hydrophobic domain(s) (Fig. 5). This is an important fact to emphasize and offers the key to an understanding of the adsorption of the enzyme onto the insoluble solid. Studies are under way to define the structure of PudA by more detailed analysis of deletions and mutations. Determination of the three-dimensional structure of PudA will contribute to the confirmation of this structual prediction and to the clarification of whether the structure of this bacterial enzyme conforms to that of the AChE which exists in eukaryotes. Neither PudA nor AChE possessed any activity of the other enzyme (data not shown). Inspection of the nonconserved regions in PudA and T AChE should also provide some important information about the regions involved in substrate recognition and catalytic activity. These analyses will go far to advance study of the molecular evolution of these esterases. ACKNOWLEDGMENT
This work was partIy supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES
1. Darby, R. T. and Kaplan, A. M.: Fungal susceptibility of polyurethanes. Appl. Microbial., 16, 900405 (1968). 2. Pathirana, R. A. and Seal, K. J.: Studies on polyurethane
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deteriorating fungi. II. Au examination of their enzyme activities. Int. Biodeterior., 20, 163-168 (1984). 3. Kay, M. J., Morton, L. H. G., and Prince, E. L.: Bacterial degradation of polyester polyurethane. Int. Biodeterior., 27, 205-222 (1991). 4. Crabbe, J. R., Campbell, J. R., Thompson, L., Walz, S. L., and Schultz, W. W.: Biodegradation of a colloidal ester-based polyurethane by soil fungi. Int. Biodeterior. Biodegrad., 33, 103-113 (1994). 5. Nakajima-Kambe, T., Onuma, F., Kimpara, N., and Nakabara, T.: Isolation and characterization of a bacterium which utilizes polyester polyurethane as a sole carbon and nitrogen source. FEMS Microbial. Lett., 129, 3942 (1995). 6. Nakajima-Kambe, T., Onuma, F., Akutsu, Y., and Nakahara, T.: Determination of the polyester polyurethane breakdown products and distribution of the polyurethane degrading enzyme of Comamonas acidovorans strain TB-35. J. Ferment. Bioeng., 83.454458 (1997). 7. Akutsu, Y., Nakajima-Kambe, T., Nomura, N., and Nakabara, T.: Purification and properties of a polyester polyurethane-degrading enzyme from Comamonas acidovorans TB35. Appl. Environ. Microbial., 64, 62-67 (1998). 8. Tanio, T., Fnkui, T., Shirakura, Y., Saito, T., Tomita, K., Kaiho, T., and Masamune, S.: An extracellular poly(3-hydroxybutyrate) depolymerase from Akaligenes faecaks. Eur. J. Biochem., 124, 71-77 (1982). 9. Mukai, K., Yamada, K., and Doi, Y.: Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei. Int. J. Biol. Macromol., 14, 235-239 (1992). 10. Jendrossek, D., Knoke, I., Habibian, R. B., Steinbuchel, A., and Schlegel, H. G.: Degradation of poIy(3_hydroxybutyrate), PHB, by bacteria and purification of a novel PHB depolymerase from Comamonas sp. J. Environ. Polym. Degrad., 1, 53-56 (1993). 11. Miiller, B. and Jendrossek. D.: Purification and properties of a poIy(3-hydroxyvalerate) depolymerase from Pseudomonas lemoignei. Appl. Microbial. Biotechnol., 38, 487492 (1993). 12. Scbirmer, A., Jendrossek, D., and Schlegel, H.G.: Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fruorescens GK13. Appl. Environ. Microbial., 59, 1220-1227 (1993). 13. Kasuya, K. and Doi, Y.: Enzymatic degradation of poly[(R)-3. hydroxybutyrate] by Comamonas testosteroni ATSU of soil bacterium. Polym. Degrad. Stabil., 45, 379-386 (1994). 14. Kita, K., Ishimaru, K., Teraoka, M., Yanase, H., and Kato, N.: Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122. Appl. Environ. Microbial., 61, 1727-1730 (1995). 15. Nojima, S., Mineki, S., and Iida, M.: Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerase s produced by Agrobacterium sp. K-03. J. Ferment. Bioeng., 81, 72-75 (1996). 16. Jendrossek, D., Schimer, A., and Schlegel, H. G.: Biodegradation of polyhydroxyalkanoic acids. AppI. Microbial. Biotechnol., 46, 451-463 (1996). 17. Bebrends, A., Klingbeil, B., and Jendrossek, D.: Poly(3hydroxybutyrate) depolymerases bind to their substrate by a Cterminal located substrate binding site. FEMS Microbial. Lett., 143, 191-194 (1996). 18. Jendrossek, D., Backhaus, M., and Andermann, M.: Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. Can.
J. Microbial., 41, 160-169 (1995). 19. Mukal, K., Yamada, K., and Doi, Y.: Kinetics and mechanism of heterogeneous hydrolysis of poly[(R)-3.hydroxybutyrate]
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film by PHA depolymerases. Int. J. Biol. Macromol., 15, 361364 (1993). 20. Yanisch-Perroo, C., Vieira, J., and Messing, J.: Improved Ml3 phage cloning quences of the Ml3
vectors and host strains: nucleotide semp and pUC vectors. Gene, 33, 103-119
(1985). 21. Maniatis, T., Fritsch, E. F., and Sambrook, J.: Molecular Cloning: a laboratory manual 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989). 22. Kay, M. J., McCabe, R. W., and Morton, L. H. G.: Chemical and physical changes occurring in polyester polyurethane during biodegradation. Int. Biodeterior. Biodegrad., 31, 209-225 (1993). 23. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685 (1970). 24. Pugsley, A. P.: The complete general secretory pathway in gram-negative bacteria. Mi&obiol. Rev., 57, 50-108~(1993). 25. Schrae.__ 3. D.. Li, Y., Wu. S.. and Cleler. M.: Ser-His-Glu -_ trian forms the catalytic site of the lipase from Geotrichum candidum. Nature, 351, 761-765 (1991). 26. Sussumau, J. S., Harel, M., Frolov, F., Oefner, C., Goldman, A., Toker, L., and Silman, I.: Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science, 253, 372-379 (1991). 27. Creler. M.. Schrag. J. D.. Sussman. J. L.. Harel. M.. Silman. Y:,- Gentry; M. K, and Doctor, B: P.: Relationship’ between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins. Protein Sci., 2, 366-382 (1993). 28. Brenner, S.: The molecular evolution of genes and proteins: a tale of two serines. Nature, 334, 528-530 (1988). 29. Schumacher, M., Camp, S., Maulet, Y., Newton, M., MacPheeQuigley,
K.,
Taylor,
S. S.,
Frledmann,
T.,
and Taylor,
P.:
Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA seauence. Nature. 319, 407-409 (1986). 30. Lockridge, Norton,
O., Bartels, C.-F., Vaughan, T. A., Wong, ‘C. K:, S. E., and Johnson, L. L.: Complete amino acid se-
quence of human serum cholinesterase. 549-557 (1987).
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31. Sbimada, Y., Sugihara, A., Bzumi, T., and Tominaga, Y.: cDNA cloning and characterization of Geotrichum candidum lipase II. J. Biochem., 107, 426-430 (1990). 32. Zock, J., Cantwell, C., Swartling, J., Hodges, R., Pobl, T., Sutton, K., Rosteck P., Jr., McGilvray, D., and Queener, S.: The Bacillus subtilis pnbA gene encoding p-nitrobenzyl
esterase: cloning, sequence and high-level expression in Escherichiu coli. Gene, 151, 37-43 (1994). 33. Lockridge, O., Adkins, S., and La Du, B. N.: Location of disulfide bonds within the sequence of human serum cholinesterase. J. BioI. Chem., 262, 12945-12952 (1987). 34. Fukui, T., Narikawa, T., Miwa, Shirakura, Y.. Saito, T., and Tomita, T.: Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. Biochim. Biophys. Acta, 952, 164-172 (1988). 35. Jendrossek, D., Frisse, A., Bebrends, A., Andermann, M., Kratzin, H. D., Stanislawski,
T., and Schlegel,
H. G.: Biochem-
ical and molecular characterization of the Pseudomonas lemoignei depolymerase system. J. Bacterial., 177, 596-607 (1995). 36. Hansen, C. K.: Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates. FEBS Lett., 305. 91-96 (1992). 37. Shinomiya, Y., Iwata, T., Kasuya, K., and Doi, Y.: Cloning of the gene for poly(3-hydroxybutyric acid) depolymerase of Comamonas testosteroni and functional analysis of its substrate-binding domain. FEMS Microbial. Lett., 154, 89-94 (1997).
Cloning and Sequence Analysis of a Polyurethane Comamonas acidovorans TB-35 NOBUHIKO
NOMURA,
YUKIE SHIGENO-AKUTSU, TOSHIAKI AND TADAATSU NAKAHARA
Institute of Applied Biochemistry,
Esterase of
NAKAJIMA-KAMBE,*
University of Tsukuba, Tsukuba, Ibaraki 305, Japan
Received 13 June 1998/Accepted 6 July 1998 Comamonas acidovorans strain TB-35 has an esterase that degrades solid polyester polyurethane (PUR). The structural gene,pudA, for the PUR esterase has now been cloned in Escherichia coli. WhenpudA was expressed in E. coli, the recombinant protein was able to degrade solid PUR. The predicted amino acid sequence contains the Gly-X1-Ser-X2-Gly motif characteristic of serine hydrolases. The highest degree of homology was detected with the Torpedo californica acetylcholinesterase (T AChE), possessing the Ser-His-Glu catalytic triad, with the glutamate residue replacing the usual aspartate residue. Similarity in the number and positions of cysteine and salt bonds was very apparent between PudA and T AChE, as were also identities of sequences and their positions in the a-helix and &strand regions between the two. In the neighborhood of the glutamate residue of the Ser1~-His@3-Glu3” catalytic domain of PudA, there were three hydrophobic domains, one of which constituted the surface-binding domain, which occurred in the C-terminus of most bacterial poly(hydroxyalkanoate)(PI-IA) depolymerases. [Key words:
Comamonas acidovorans, polyurethane, esterase, cloning, nucleotide sequence]
Polyurethane (PUR), which is a kind of plastic, is widely used as a base material in various industries. PUR is an artificial synthetic polymer which is waterinsoluble (hydrophobic), and is generally synthesized by condensation of polyol and polyisocyanate. An estertype PUR degradation activity has been reported in several microorganisms, but the enzymes have not yet been purified, or characterized in detail (l-4). In our previous papers, we reported the isolation of a bacterium, Comamonas acidovorans TB-35, which could utilize solid polyester PUR as the sole carbon and nitrogen source (5). We also found that an esterase played a major role in PUR degradation by this strain (6). This strain had two kinds of extracellular esterases; one secreted into the culture medium and the other, bound to the cell surface. Only the cell-bound esterase could degrade PUR. Recently, we succeeded in purifying the cell-bound esterase (PUR esterase) and reported that this enzyme probably degrades PUR in a two-step reaction; hydrophobic adsorption onto the solid PUR surface and hydrolysis of the ester bonds of PUR (7). Regarding solid polyester biodegradation, it is well known that poly(3-hydroxyalkanoate), PHA, can be degraded by a large variety of microorganisms (8-15). The key enzyme for PHA degradation, PHA depolymerase, is also a kind of esterase. Almost all PHA depolymerases consist of two separate domains, the catalytic domain and the PHA surface-binding domain (16-18). It has been reported that the structure of the PHA depolymerase is such that inhibition of the degradation activity occurs in the presence of excessive amounts of the enzyme (19). However, we recently demonstrated that the degradation activity of the PUR esterase (PudA) did not decrease but rather remained constant when an excess of the enzyme was present (7). It was also indicated by enzymatic characterization that the PUR esterase was a novel plastic-degrading esterase
with properties different from those of lipases or PHA depolymerases. In this study, we present the molecular cloning and sequence analysis of the PUR esterase gene in C. acidovorans TB-35, and its functional expression in E. coli. By sequence comparison, it was shown that the amino acid sequence of the PUR esterase was similar to that of the Torpedo californica acetylcholinesterase (T AChE), and very different from that of the PHA depolymerases except in respect of the PHA surface-binding domain. The putative structure and characteristics of the PUR esterase encoded by the gene are discussed. MATERIALS
AND METHODS
Materials The polyester polyurethane (PUR) used in this study was synthesized by the condensation of poly(diethylene glycol adipate) (DEGA) and 2,4-tolylene diisocyanate under anhydrous conditions as described previously (6, 7). The DEGA was supplied by Suzuki Motor Co. (Hamamatsu). E. cofi DHlOB was purchased from Toyobo Co. Ltd. (Osaka). All other compounds used were standard commercial preparations. Bacterial strains, plasmids, and media The culture conditions for C. acidovorans TB-35 have been described previously (5). E. coli DHlOB and JMl09 were used for the isolation and maintenance, respectively, of recombinant derivatives of pUC19 (20). E. coli strains were grown at 37°C on Luria-Bertani (LB) medium (21) with appropriate supplements. Ampicillin (final concentration of 100 pg/ml) was added to the medium, when necessary, for plasmid selection. The expression of the PUR esterase gene in recombinant E. coli strains was tested on an indicator plate. The plate was prepared by pouring an overlay of 1.5% agar solution containing a sonicated emulsion of 0.5% DEGA onto a previously prepared LB agar plate (7). Enzyme activity assays The esterase activity for a water-soluble substrate (p-nitrophenyl acetate) was deter-
* Corresponding author. 339
340
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ET AL.
mined by the method of Kay et al. (22). One unit was defined as the amount of enzyme required to liberate 1 /*mol of p-nitrophenol per min. The PUR degradation activity was determined by the procedure described previously (6, 7). Samples (0.5 ml each) of 1OOmM potassium phosphate buffer (pH 7.0) which contained cell-free extracts of recombinant E. coli culture media were transferred to small test tubes. The reaction was started by adding a PUR block (4 x 4 x 1 mm) to each of the tubes. After 48 h incubation at 3O”C, the PUR blocks were removed, and the amount of free diethylene glycol produced by hydrolysis of the ester bond, was quantified by gas chromatography (GC) using a model GC-8A gas chromatograph (Shimadzu Co., Tokyo), as described previously (6). Preparation of the cell-free extract from recombinant One loopful of cells of recombinant E. coli E. coli JM109 was inoculated into a 5OOml shaking flask containing 1OOml of LB medium (100 /*g/ml ampicillin). After incubation for 5.5 h at 37”C, cells were harvested by centrifugation at 8000 x g for 10 min. The cells were suspended in 20 mM potassium phosphate buffer (pH 7.0) and disrupted using a sonicator. The disrupted suspension was centrifuged at 12,000 x g for 20 min to remove cell debris and unbroken cells. Protein SDS-PAGE analysis was Protein analysis performed as described by Laemmli (23). Proteins were electroblotted from SDS-polyacrylamide gels onto a polyvinylidene difluoride membrane (PVDF) (Clear Blot Membrane-P, ATT0 Co. Ltd., Tokyo) and the N-terminal sequence was determined using an automated protein sequencer (model 492, Parkin Elmer Applied Biosystems, Tokyo). General molecular procedures and computer analysis Unless specified otherwise, standard methods for DNA manipulation described by Maniatis et al. (21) were followed. Southern hybridization was carried out using hybridization kits (ECL 3’-Oligolabelling and detection system, Amersham, London, UK). An ABI PRISM Dye Terminator Cycle sequencing Core kit (Parkin Elmer Applied Biosystems) was used for DNA sequencing. The products of the termination reactions were analyzed with the aid of an automated DNA sequencer (model 373A, Parkin Elmer Applied Biosystems). Sequence comparisons were made using the EMBL and GenBankTM databases. The nucleotide and amino acid sequences were analyzed using the GENETYX-MAC program, version 8 (Software Development Co. Ltd., Tokyo), FASTA (ver. 3.0), and BLAST (ver. 2.0). The predicted secondary structure of PudA was calculated by the DDBJ SSTHREAD program. The nucleotide sequence data reported in this paper can be obtained from the DDBJ nucleotide sequence database with the accession number AB009606.
entire genome of C. acidovorans TB-35, completely was hybridized with the digested with EcoRI, oligonucleotide probe on a Southern blot, to produce hybridized fragments of about 10.0 kbp (data not shown). Fragments with a size of about 10.0 kbp were recovered from the agarose gel and ligated with the EcoRI-digested and dephosphorylated vector, pUC19. The ligation mixture was used to transform E. coli DHlOB. About 50 recombinant clones were screened by Southern hybridization of the EcoRI-digested plasmid DNA using the oligonucleotide as a probe. A 10.0 kbp EcoRI-digested plasmid DNA fragment from each clone specifically hybridized with the probe and indicated that the 5’ region of the PUR esterase had been cloned. The clones expressed esterase activity in E. coli and produced a translucent clear zone on an indicator plate containing DEGA. To define the location of the gene on the 10.0 kbp fragment that hybridized with the probe, the fragment was digested with restriction enzymes. The fragments were subcloned into the appropriate sites of the pUC19 vector. The resultant plasmids were tested for their abilities to produce clear zones on an indicator plate. The gene responsibIe for clear zone formation was located in pUR22, which had the smallest length of DNA, of less than about 2.2 kbp. Functional expression of p&A gene in E. coli To investigate the biochemical properties of the recombinant PUR esterase, the protein was expressed in an E. coli strain carrying pUR22. The recombinant protein separated by SDS-PAGE is shown in Fig. 1. It was observed that the apparent molecular masses of both the recombinant PUR esterase and the native PUR esterase purified from C. acidovorans TB-35 were identical. No relevant band was observed at the same mobility in the lane containing the extract from E. coli carrying pUC19. The recombinant PUR esterase was tested for its ability to degrade PUR. The esterase activity of the cell-free extract of the recombinant E. coli carrying pUR22 was 0.3 U/mg protein. The PUR cube was degraded by the extract producing diethylene glycol (50 pmol/mg protein/48 h), a degradation product which was detected by GC analysis. The cell-free extract of E. coli carrying pUC19 did not possess the esterase activity and degrada-
kDa
1
2
3
4
RESULTS Cloning of the gene encoding the PUR esterase Previously, we purified the cell-bound esterase, a PUR degradation enzyme, from C. acidovorans TB-35 (7). The amino acid residues at the N-terminus of the PUR esterase were sequenced (N-(C)GGSDNDSSSNNQGAPA VA-C). An oligonucleotide (5’AAC GA(G/C) AGC TCG AGC AAC AAC CAG GGC-3’) was synthesized using 9 amino acids (NDSSSNNQG) present in the N-terminal 19 amino acids of the PUR esterase taking into consideration Comamonas species codon preferences (18). The
20.1 14.4 FIG. 1. SDS-PAGE analysis of the crude extract from E. co/i containing pUC19 or pUR22, respectively (lanes 2 and 3), and of the purified esterase from C. acidovorans TB-35 (lane 4). The sizes in kilodaltons of the molecular weight markers (lane 1).
CLONING
VOL. 86, 1998
OF COMAMONAS
POLYURETHANE
-339
GTCGCAAGGCGAGCCGGATTGGGATCTGGCAGCTCT~CAGCTC~CCCGACGA~TAGACCAGCGCGTC~TT~TGACCCAGTGA
-261
AGCGGCGGTATCCATCGCTGCGGGGAAGCAGCTGCGCGCGCGCGCGCCAGGCCC~TGCATACT~CTCCC~GCTCTTGC
ESTERASE
GENE
341
-262 -184
-35 -183
CATT(;AGCGGCAAGCGAGCGCTCAACCTTCCTTCCGACCT
-106
-10 -105
-27
+PUa ATACl3AAGATCCTTC~ATTG~TGAATGAATAGCCGTTCCCT~CT~GCGATCCGATTTCCCAC~TTCTTGCA MNSRSLTKAI
S.D.
52
F
129
SSNNQGAPA 207
RLGMRRYFG
TVAGQVQAVD
I
P
F
A 285
GCGCCTCCGGTGGGCAACCTGCGCTGGATGCCGCCGCCGGCGCCGCCTC~TCCTGGGCCGCCCCGCTGGCG~GACCCAG 4
286
SVLGACGGSDNDS
GTTGCCATTACGGTCGCAGGCCAGGTCCAGGCTGTGTGGACCGCCTCGGCATGCGCAGATATTTCGGGATACCTTTTGCC VAI
208
51 RFPTILA
CTCGCCGGCTTCAGCGTACTTGGTGCATGCGGTGGCAGCGAC~CGACAGCAGTAGT~C~CCAG~CGCACCCGCC LAG
130
-28
CATGCGGTTGAATTTCCTATCCGTGGTAGCATCGTTCGCATCGTTCGCGCGG~T~T-C~CATTTCGCA~GT-CAC
P
PVGNLRWMPPAPPQSWAA
PLAKTQ 363
TCTAATGCGCCCTGCATGCACCGGCGCCACCGATCCCCTCCGCCTGCCC~CGGCACCGA~ACTGCCTCTACCTC SNAPCMQTGATDPLRLPNGTEDCLYL
364
441
~ACGTCCATGCCCCTGCCACCGGCGAAGGCCCCCTTCCCTGTCATGGTGTGGATCCACGGC~CGCCTT~AGCATCGGC DVHAPATGEGPFPVMVWIHGGAFSIG
442
G 520
519
GGCACCATCACCTATGCCGACCCGTCGCCCCTGGTCAGCAGGGC T
PLVSKGVIVVN
ITYADPS
IAYRMG 597
GCCATGGGTTTCCTGGGCCATCCGTCGCTGCGCGCCGCGCACCACGGTC~C~CTACGGCATCAT~ACCAGCAG AKGFLGHPSLRAADGTVGNYGIMDQQ
598
A 676
WV
Q
D
N
IAAFGGDKSNVTI
F
C:
G
F
753
ci
YGFDRQ
LTQAQL
E
A
Q
S
A
A
G
V
S
C
AT
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F
T
TAN
W
S
P
V
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S
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G
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K
987 S
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K 1065
DEWSYFV
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RELVAGPLTAAQYPSYLQTS
1143 G
L
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8:
Y
1221
1299 SQATPIF
TDMHFSCPALNLSKRVL
E
L
1377
SFNQGAGH
IGRNTI
Q
YLFNLRDLETAEHRDLQA
1455 S
MA 1533
CGCTACTGGACCAACTTTGCCCGCACCAGCAATCCGAATATTACG SWPAFT
YWTNFARTSNPNNGDPVAT
1611
GGACCGACCAAGGTGCTCGGACTGGACGTCGCCTCCGCTGGCGGCATCAG~~CTGGCCACGTTCGAGACCGACCAC IRELATFETDH
PTKVLGLDVASAGG
1689
AAG'TGCAACACGGCCTGGACATCACTGACTTTTTGATCACCTTTTT K
':
NT
A
P
SLAATA
LATVYPLTDYGTNTAQQP
ACGTATGAGCTCCAGTACCTGTTCAACCTGCGTGACCTGCGTGACCTGGAGACCGC~~CACCGCGACCTGCAGGCCAGCATGGCA
G 1612
K
ATGTACGAGTTCCGGGACCGGACTGCGATCCCGTCGATCGGCCGC~CACTATCAGTTTC~CCAG~GGCAGGTCAC
R 1534
909
GCC';GCACCGACATGCACTTTTCCTGTCCGGCGTTGAACTCTTC
T 1456
831
IVNSAL
PTVDAACLRGLSAELVNNQ
MYEFRDRTAIPS 1378
S
CCA'rCGCTCGCCACGGTATATCCACTGACGGATTACGGCACC~TACCGCGCAGC~CCCAGCCTC~GGCGACGGCG
A 1300
T
GCATCGCGCGAACTGGTGGCGGGGCCGCTGACCGCTGACCGCTGCCCAGTACCCTTCCTATCTGCAGACCTCGCTCGGTCTGCCA
P 1222
S
GCGACTTTCGTCGCCGGGGAGAACAACAAGGTGCCGCTGGTTCGTT
A 1144
E
PLSKGLFAKAIVQS
SVMTHLAS
ATFVAGENNKVPLVNGSNQ 1066
G
CTCGCGACCGCATTCACCACGGCCRACTGGAGCCCCGTGGAGCC~CGTGCCTTCGGT~ATGGC~GGTTTTGCCC~GTCCATC~G L
988
R
GCCOCAGCCGGCGTTAGCTGCCCCACCGTCGACGCGGCATGTTTGCGCGGCTTGTCCGCCGAGCTTGTC~C~CCAG A
910
L
GGCC:GCTACGGATTCGATCGCCAGCTGACACAGGCGCAGCTTG~GCGCAGAGCACGTCGATCGTC~CAGCGCGCTG G
832
A
GCCGGCGGCTTCAGCGTGATGACACACCTGGCGTCACCGCGC A
754
675
GCAGCACTGCGCTGGGTGCAGGACAATATCGCCGCGTTCGCCGCGTTCGGCGGCGAC~GTCC~TGTCACCAT~TTCGGCG~TCC
W
T
S
L
T
F
dt--
1690
TATTGCACCGCATGACGTTTGGGACGCAAGTCATTCATTCCTTC~GATACTCGCGCAGTC~CCCT~ACCATTTCCAGTT
1768
CCT~ACACACCAGATGAGACCGTTGGTTTTCTCTGGCAAGCATGC
1767 1835
FIG. 2. Nucleotide sequence of PUR &erase. The deduced amino acid sequence is shown under the nucleotide sequence. A putative promoter region, Shine-Dalgano sequences (S. D.), and p-independent terminator are indicated. The chemically determined N-terminal amino acid sequences of the PUR esterase purified from C. acidovorans TB-35 are indicated with a single underline.
342
NOMURA
ET AL.
J. FERMENT. BIOENC.,
tion activity of PUR. These results indicate that the gene encoding for the PUR esterase is included in the DNA fragment inserted in pUR22. Nucleotide sequence and deduced product of the DNA that encodes the PUR esterase The nucleotide sequence of the 2174 bp DNA fragment inserted in pUR22 was determined for both strands. The complete nucleoS
tide sequence of the sense strand and the deduced amino acid sequence are shown in Fig. 2. Computer analysis of the nucleotide sequence detected one open reading frame (ORF), located between nucleotides 1 and 1644. The molar G+C content was 63.4%. At the third position of the codon, G or C was found in 78.9% of the cases. The ORF consists of 1644 bp with a putative ATG initiation h-
S-
s
MPPAPPQSWAAPL-AKTQSNAP ** l
PUdA
1:CGGSDNDSSSNNQGAPAVAITVAGQVQAVDRLGMRRYFGIPFAAPPVGN ***** * * *
T AChE
l:-----DDHSELLVNTKSGKV-MGTRVPVLSG-HISAFLGIPF~PPVGN~R~EPKKPWSG~AST~N-~QYVD
s-s
-9
*****
w
72
s-
3
L
s
b
9,
PUdA
77:---TG-ATDPLRLPN-G*
YLDVHAPATGEGPFPVHVWIHGGAFSIG-GTI-TYADPSPLVSKGVIWNIA ***** ** * * * * *
T AChE
73:EQFPGFSGSEMWNPNREM
YLNIWVPSPRPKSTTVMVWIYGGGFYSGSSTLDVYNGKYLAYTEEWLVSLSY s-s
e-h
s-
h-s-
PudA T AChB
153:FGFL---ALHGS
I -h
II
h-h
PUdA
2 2 7 : GYGFDRQLTQAQLEAQS
T AChE
228:SPNCPWA-----SVSVAEGRRRAVELGRNL
--
2 99 KKPQELIDVEW’NVLPFDSIFRFSFVPVIDGEFFP
h
M-h
h
302
h-h
III h PUdA T AChE
h
+
s-s
h-h
300:KSIKATFVAGENNKVPLVNGSNQDEWSYF-V-ASRE l * * * * l * * *
**
h-h
s-s
h
*
h-
h-
T AChE
h *
**
-GTN *
303:TSLESMLNSGNFKKTQILLG~KD~GSFFLLYG~GFSKDSRSKISR~DFMSG~LS~HANDLGLDA~LQYTD~DDN -h
PUdA
h
h
382
h-
+
S---8
371:TA-QQP-SLAATAAGTDMHFS *
~NLSKRVLSQATPIFMYEFRDRTAIPSIGRNTISFNQGAGHTYELQYLFN--L-RD * ** ** ***
383:NGIKNRDGLDDI-VG-DHNVI
LMHFVNKYTKFGNGTYLYFFHR-A-SNLVW-P-EW-MDV~ h
h
445 *
l
455
s-
-h
h-
S----8
522
PudA * T AChE
370
***
*
**
**
*
***
**
**
*
*
456:LNYTAgEE~SRRIMHYWATF~TGNPNEPHSQESK~LFTTKEQKFIDLNTEPMKVHQRL----RVQ h S” sh
FWNQFLPKL h
531
h
PudA T AChE
532:LNATACDGELSSSGTSSSKGIIFYVLFSILYLIF
FIG. 3. Comparison of the (26). Residues that are identical Regions 1, II, and III are shown under the sequence, respectively. Cys-Cys bridges and conserved
amino acid sequences of C. acidovoruns PUR esterase (PudA) and T. californicu acetylcholinesterase (T AChE) in both sequences are indicated by an asterisk (single-letter amino-acid notation). Gaps are indicated by hyphens. in black boxes. The secondary structure elements observed in PUR esterase and T AChE are shown above and a helix is shown as h-h; ,9 strand is shown as s-s; + indicates residues from the catalytic triad; the pattern of salt bridges are also shown by boxes.
CLONING OF COMAMONAS
VOL. 86, 1998 codon
that is 7 bp downstream from a potential ribosome-binding site (~ 12AGGAGA-7). No other putative start codons were found in the nucleotides upstream. Examination of the nucleotide sequences for the presence of a p-independent terminator of transcription revealed a stem and a loop followed by a short stretch of thymidine residues (1667GGCCCGC----GCGGGCCTT TTTTATTL693). A potential promoter sequence similar to the -35 and ~ 10 consensus sequences of E. coli was found upstream of the ORF, although the transcription start site has not yet been mapped. The nucleotide sequence of the ORF coded exactly for the chemically determined N-terminal amino acid sequence ((C)GGSD* l) of the purified PUR esterase from C. acidovorans TB-35 (Fig. 2). The ORF for the PUR esterase contained a putative start codon, 26 amino acids upstream of the experimentally determined N-terminal codon. The ORF encodes 522 amino acid residues capable of forming a 55,110 Da processed protein. It was concluded that this ORF coded for the PUR esterase and was named pudA. Analysis of the deduced amino acid sequences of pudA The first 26 deduced amino acids showed the characteristics of signal peptides of secretory precursors; three positively charged amino acids, arginine at positions 4 and 11 and lysine at position 8, were followed by a core of mainly hydrophobic amino acids (Fig. 2). The mature PUR esterase from C. acidovorans TB-35 has the sequence (Cys’)-Glyz-Gly3-Se&Asps at the N-termini. Ala-’ is a well-known cleavage site for known leader peptidases (24). From computerized searches of databases, it was shown that PudA possessed a high degree of homology only with the catalytic regions of the serine hydrolase family proteins which contain the Ser-His-Glu catalytic triad with a glutamate residue replacing the usuai aspartate residue (25-28) (Table 1). Comparison of the positions of each residue of the Ser-His-Glu catalytic triad showed that the amino acid residues of PudA were in similar positions to those in the Torpedo californica acetylcholinesterase (T AChE) and the human cholinesterase (H ChE). In particular, PudA is 30.1% identical to T AChE (Fig. 3). Comparison of the positions of Cys residues in PudA and T AChE showed that the disulfide bonds were in identical positions (positions 74-92, 258-265, and 390-513). We carried out the calculation of the secondary structure of PudA by computer analyses and the result was compared with that of T AChE which was already determined by three-dimensional analysis (26). It was shown that the number and positions of the a-helix regions and j-strand regions in PudA were similar to those in T AChE (Fig. 3). Predictable identification of the PUR surface-binding domain We have already demonstrated, by enzymatic characterization, that the PUR esterase possesses highly l
POLYURETHANE
ESTERASE GENE
343
Region II I of PudA xi9
321 ~~QDEWSYFVASRELVAG-PLTAAQY~SYLQTSLGLPPSLATXJY~LTDPGT . ...** * *. . .*.* .*..*.* ***. . . ATvTNHYVAGRINVTQYNvLGARY-GYVl'TPLYYCPSLSGwTDKANCPI
PhaZi p/e PHAsurface-binding domain FIG. 4. Comparison with amino sequences of the PHA surfacebinding domain. Sequence alignment of C. acidovoruns PUR esterase (PudA) and the PHA surface-binding domain of the PHA depolymerase (PhaZlpk) (18). Identity and similarity of sequences are shown as * and , respectively. hydrophobic region(s) and that the hydrophobic adsorption of these region(s) onto the surface of the PUR is important for PUR degradation (7). Analysis of the primary structure of PudA revealed that PudA possessed three regions (region I, II, and III) containing many hydrophobic amino acid residues, in the center of its polypeptide chain (Fig. 3) and that the amino acid sequence of one (III) of the three regions was highly homologous to that of the PHA surface-binding domain of PHA depolymerase (PhaZlple) of Pseudomonas femoignei (Fig. 4) (18). No identity or similarity between PudA and PHA depolymerases was observed except in the PHA surface-binding domain. The three regions in this aforementioned domain include many hydrophobic amino acid residues different from those in T AChE which cannot degrade PUR. The hydropathy profiles for PudA and T AChE were similar except in the three regions (I, II, and III) (data not shown).
DISCUSSION We have cloned the structural gene (PudA) encoding the cell-bound PUR esterase from C. acidovorans TB35, and analyzed its sequence. It was demonstrated that recombinant PUR esterase could be expressed in E. coli as an active protein for the degradation of solid PUR. The amino acid sequence of pudA revealed no significant homology to sequences of PHA depolymerases except within the PHA surface-binding domain. PudA has regions similar to those of proteins in the family of serine hydrolases which possess the Ser-His-Glu catalytic triad (Table 1). Although alignments of PudA never showed identity of more than 30.1% (T AChE) in amino acid sequences, the sequence similarity between PudA and T AChE extends throughout the complete amino acid sequence of these proteins, being especially pronounced in some regions with a high degree of conservation. Also, the positions of Cys residues in PudA and T AChE were similar (Fig. 3). In T AChE, the sequence includes three pairs of Cys residues (position 67-
TABLE 1. Aligment of amino acid sequence of PUR esterase with the sequences of four Ser-His-Glu type serine hydrogenases Serine (S)” PudAb
T AChE HChE CCL1 PnbA Consensus
199 200
198 218 167
Glutamate (E)
FGGDKSNVTIFG~SAGGFSV FGGDPKTVTIFGESAGGASV FGGNPKSVTLFGESAGAASV FGGDPDKVMIFGESAGAMSV
324 327 325 354
GSNQDmSYF GVNKDEGSFF GVNKDEGTAF GNQEDEGTAF
FGGDPDNVTVFG~SAGGMSI FGGdp--Vt*FGl#SAG*-Sv
288
GTTRDZGYLF G---DEg*-F
Histidine (H) 433
440 438
GAGHTYELQYLF GVIBGYEIEFVF GVMHGYEIEFVF
463 377
GTFHGNELIFQF KAFEALELPFVF
g--H--E*_f-F
a The positions of serine, glutamate and histidine in the primary sequences of the corresponding mature proteins are indicated. b PUR esterase from C. acidovorons TB-35 (PudA), acetylcholinesterases of T. californicu (T AChE) (29), cholinesterase of humans (H ChE) (30), Geotrichum cundidum lipase isoform1 (GcLl) (31), p-nitrobenzyl esterase from Bacillus subtifis (PnbA) (32).
344
NOMURA ET AL.
J. FERMENT. BIOENO.,
-2
PudA
-21 1
200
T AChE
n
I
E 327
1
I
H440
1
.
I
-3
PhaZl me
PhaZppi
B
zie ;;;m$ne-rich
1
Lipase box
a
;;M;;;;;dule
l l
of
Cysteine
m
Disulfide bond
;;/;;rface-binding
II
FIG. 5. Comparison of the domain structures. C. ucidovorum PUR esterase (PudA), T. culifornicu acetylcholinesterase bacterial PHA depolymerases (PhaZple and PhaZppO (18, 35).
94, 254-265, and 402-521) that were found to participate in the stable formation of intra-molecular disulfide bonds (33). Between PudA and T AChE structures, the identities (or the similarities) and positions of sequences are also very apparent in the a-helix regions, p-strand regions, and salt bonds (Fig. 3). This suggests that the protein folding and the secondary structures of these enzymes are similar and therefore also share a common three-dimensional fold. Almost all PHA depolymerases consist of two separate domains, the catalytic domain and the PHA surface-binding domain (17, 34) (Fig. 5). These domains are linked by a flexible linker, which is a threonine-rich region (35) or a fibronectin type III-like module (Fn3), which is a eukaryotic extracellular matrix protein (36). By the previous biochemical characterization of the PUR esterase, we hypothesized that the surface-binding site and catalytic site of this enzyme existed nearby in three-dimensional space, unlike those of the PHA depolymerase (7). It was estimated here that PUR esterase included three putative surface-binding domains including many hydrophobic residues in the center of the enzyme polypeptide chain (Fig. 5). As an enzyme that possesses a structure that resembles PudA, the structure of T AChE differs from that of the PHA depolymerases in the catalytic and binding domains constructed by three-dimensional analysis (26). The flexible linker domain was also not observed in PudA and T AChE (Fig. 5). By these estimations and the remarkable structural similarities with T AChE, it is concluded that the structure of PudA is not similar to that of the PHA depolymerases and that the three putative binding domains of PudA are positioned close to the catalytic domain in three-dementional space. The conclusions support our hypothesis based on the enzymatic characterization of the PUR esterase. It would seem that PudA and the PHA depolymerases have evolved from distinct origins because of the differences in the structures of both enzymes. PUR esterase and PHA depolymerases are of interest primarily because of their structures facilitating the adsorption of insoluble solids. It was clearly demonstrated
(T AChE) (26), and
that a hydrophobic domain (surface-binding domain) of the C-terminus of a PHA depolymerase binds to solid PHA (37). We also demonstrated that the purified PUR esterase has hydrophobic region(s) and that the hydrophobicity is important for the adsorption onto PUR. In this study, it was shown that PudA possesses three domains which contain many hydrophobic residues and region III of the three hydrophobic regions of PudA is similar to the PHA surface-binding domain of the PHA depolymerase. It was concluded that all or some of the three regions could play a role in the binding of PUR. Although the structures of PudA and the PHA depolymerases are probably be very different, both enzymes possess hydrophobic domain(s) (Fig. 5). This is an important fact to emphasize and offers the key to an understanding of the adsorption of the enzyme onto the insoluble solid. Studies are under way to define the structure of PudA by more detailed analysis of deletions and mutations. Determination of the three-dimensional structure of PudA will contribute to the confirmation of this structual prediction and to the clarification of whether the structure of this bacterial enzyme conforms to that of the AChE which exists in eukaryotes. Neither PudA nor AChE possessed any activity of the other enzyme (data not shown). Inspection of the nonconserved regions in PudA and T AChE should also provide some important information about the regions involved in substrate recognition and catalytic activity. These analyses will go far to advance study of the molecular evolution of these esterases. ACKNOWLEDGMENT
This work was partIy supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES
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VOL. 86, 1998
CLONING OF COMAMONAS POLYURETHANE
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