Biochimica et Biophysica Acta, 1174 (1993) 79-82 79 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90521 Short Sequence-Paper

Cloning and sequence analysis of an esterase gene from Pseudomonas sp. KWI-56

Yuji Shimada a Toshihiro Nagao a Akio Sugihara a, Taro Iizumi b Toshifumi Yui c, Koichi Nakamura b, Tetsuro Fukase b and Yoshio Tominaga a

a Osaka Municipal Technical Research Institute, Osaka (Japan), b Kurita Central Laboratories, Kurita Water Industries Ltd., Kanagawa (Japan) and c Department of Materials Science, Faculty of Engineering, Miyazaki University, Miyazaki (Japan)

(Received 16 March 1993)

Key words: Esterase; Nucleotide sequence; Cloning; Sequence analysis; (Pseudomonas)

The gene encoding an esterase from Pseudomonas sp. KWI-56 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide comprising 262 amino acids, whose molecular weight agreed well with the value obtained by SDS-PAGE. Comparison of the amino acid sequence with those of the other homologous enzymes suggested that Ser-92 and His-241 might be included in the catalytic triad.

Carboxylesterase (EC 3.1.1.1) catalyzing the hydroly- sis of carboxylic esters is widely distributed in animals, plants and microorganisms [1]. The enzyme from mi- croorganisms has attracted much attention, because it has become available for the production of flavor in foodstuffs and for the optical resolution of chiral sub- stances. Potent esterase-producing microorganisms in- clude Aspergillus niger [2], Ochrobactrum anthropi [3], Bacillus subtilis [4], B. stearothermophilus [5,6] and Pseudomonas fluorescens [7]. We have recently found that Pseudomonas sp. KWI-56 (KWI-56) also produces several intracellular esterases. The strain is known to secrete a thermostable lipase [8,9]. In this paper, we describe the cloning and nucleotide sequence of an esterase from KWI-56.

Esterase activity was assayed as follows: the reaction mixture containing 0.5 ml of methyl propionate (Wako Pure Chemical Industries), 2.5 ml of 50 mM phosphate buffer (pH 7.5) and 30-100/.~1 of enzyme solution was incubated at 20°C for 30 rain with magnetic stirring at 250 rpm. The reaction was stopped by adding 20 ml of ethanol, and the released acids were titrated with 50 mM KOH. One unit of esterase activity was defined as the release of 1 /xmol of acid per min. The extraction

Correspondence to: Y. Shimada, Osaka Municipal Technical Re- search Institute, Morinomiya, Joto-ku, Osaka 536, Japan. The sequence data reported in this paper have been submitted to the D D B J / E M B L / G e n B a n k Data Libraries under the accession num- ber D14529.

of chromosomal DNA from KWI-56 and DNA manip- ulation were done as described previously [9,10].

A library was constructed in the BamHI site of pUC18 with KWI-56 chromosomal DNA digested par- tially with Sau3AI and size-selected for 2-5 kbp. Among 10 000 transformants of Escherichia coil HB101,

pPE5 pUCI8

pPESsc pUCl8

pPESsp pUCl8 i ,P ~ I

pPE5sl pUClS I .~f I

PPE5e pUC]8

I ,~1 I

I

I I

I I

I I

I I I

II

Est +

Est +

Es t +

Es t ÷

E s t -

pPE5r ~ ~ o~- E

, ,[i ~ ' Est-

I i

500 bp Fig. 1. Restriction map of pPE5 and location of est. Single and double lines show pUC vectors and chromosomal DNA segments from KWI-56, respectively. Arrows indicate the location and orienta- tion of lacZ promoters on pUC vectors. Est +, production of es- terase; Est , non-production of esterase. Abbreviations for restric- tion sites are as follows: B, BamHI; Su, Sau3Al; P, PstI; Sm, Smal;

E, EcoRI; SI; SalI; Sp, SphI; Sc, Sacl.

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eight clones forming a clear zone on the tributyrin agar plate [11] were isolated. These clones were cultivated at 37°C in L-broth containing 100/xg/ml ampicillin for 16 h, and esterase activities in the cell extracts were measured. A clone which produced the largest amount of esterase (60 U / m l culture) was selected and further analyzed. A plasmid with 1.8 kbp of DNA insert was isolated from the clone and designated as pPE5. Its restriction map is shown in Fig. 1.

The total proteins produced by E. coli carrying pUC18 or pPE5 were analyzed by SDS-PAGE which was done on a 15% separation gel under the conditions developed by Laemmli [12] (Fig. 2). A protein of about 28 kDa, which was absent in E. coli HB101 [pUC18], was present in large amounts in E. coli HB101 [pPE5], suggesting that the subunit molecular weight of the esterase was 28 kDa.

To find out the orientation of the esterase gene (est) on 1.8 kbp DNA fragment, the DNA insert was rejoined into pUC19 and its direction for lacZ pro- moter was changed. The E. coli HB101 transformant with the resulting plasmid, pPE5r, did not produce esterase (Fig. 1), suggesting that est from KWI-56 expressed under the lacZ promoter on pUC vector. Subcloning experiments showed that est was located between B a m H I / S a u 3 A I and SalI (Fig. 1).

The 1.8 kbp DNA insert of pPE5 was rejoined into puCl l9 in both directions, and the resulting plasmids

1 2 3

170 kDa

97

55

37

20

Fig. 2. Esterase produced by recombinant E. coli. E. coli HB101 carrying pPE5 (lane 1) or pUCI8 (lane 2) was cultivated at 37°C in L-broth containing I00 t zg /ml of ampicillin for 16 h. Cells collected from 0.5 ml of each culture broth were heated at 100°C in 100 /xl SDS denaturat ion buffer, and the resulting samples (5 tzl) were applied to SDS-PAGE. Lane 3, Marker proteins; trypsin inhibitor (20.1 kDa), lactate dehydrogenase (36.5 kDa), glutamate dehydro- genase (55.4 kDa), phosphorylase b (97.4 kDa) and macroglobulin

(170 kDa).

-384 -360 TCTGCTTGGGTTGTAACGGGCGCGATTCGT -270 GAGCGATCGATTGCGCTGTTGGATCTCCAG -180 TGCGGCGCAGTGGATTGAAAGAGTGTCGAT

-90 TTCAATTTAAATTTGATCATCAATTATTGG

1 ATGATTTTTCATAACGGCAATGTGAATCTA 1MetIlePheHisAsnGlyAsnValAsnLeu

91 AAATCGATGTGGGATGGGTTGCGTCAAGAG 31 LysSerMetTrpAspGlyLeuArgGlnGlu

181 TGCGACCAACCCTATACGATGCTCGATCTT 61CysAspGlnProTyrThrMetLeuAsDLeu

271CATTCTCTGGGGGGGATGATCGCCCAGGAA 91 HisSerLeuGlyGlyMetlleAlaGlnGlu

361 ATCGATAACTACATGCGATCGGTAATAGAG 121 lleAspAsnTyrMetArgSerValIleGlu

451 TTTCTGGCCCTGGGTTCCAAGGCCTTGGGT 151 PheLeuAlaLeuGlySerLysAlaLeuGly

541GCGCTTATTCGCCAATGGGAGATCGATCTG 181 AlaLeulleArgGlnTrpGlulleAspLeu

631AGCGAGGATAAGATCGTGACGAAAGATCAG 211 SerGluAspLyslleValThrLysAsDGln

721CATTTTCCGATGATCGAAGCGCCGGAAGAA 241 HisPheProMetlleGluAlaProGluGlu

811 TCAATATCAACATCAATGGCGCAACGCATG 901CCGGCACGAAGTTCGGGTGCGGGATCGCGC 991 TCGCAGCCGTCCGAGACAAGAAGATAACGA

1081 TC

TCGCGTAAAGCGCTCCGGGATTGCTTGCCG AAACTCGAACCATTCCGATTTCACTGTTGT GGGTGAAGCCGATCGCAGAAAAATATTGCA CGGTTGCTGTGGATAAATATCGATCCGCCA

TCTTACGACGTGGCTGGGCATGGCGAGTGT SerTyrAspValAIaGIyHisGIyGIuCys

CTATCCGGTAAATATAGAACCGTCGCATTC LeuSerGlyLysTyrArgThrValAlaPhe

GCGAAAGATGCGCTCTCCGTGATGGACGCC AlaLysAspAlaLeuSerValMetAspAla

CTCGCGATACTGGCACCTGATCGCGTATCG LeuAlalleLeuAlaProAspArgValSer

CTTGCCCGGGACTGGAGCAAGACGATAACG LeuAlaArgAspTrpSerLysThrlleThr

TCCGATATTTTCAATCAAGTTGTGGATTTC SerAspllePheAsnGlnValValAspPhe

ACTGTCGATACAACAGATCGATTGTCGCTT ThrValAspThrThrAspArgLeuSerLeu

CAGAAAATGTTGGTAAACGGAATTTCGGGC GlnLysMetLeuValAsnGlylleSerGly

TTCATTCGAGTTTTGTCTGGCTTTATTGAC PhelleArgValLeuSerGlyPhelleAsp

GCGTCGACGTGCCGGTCGACATGCCGCTCC AGTGTGGCGCATGCACGGTGCATCTCGACG CCATCGAGGCGGTCGGCGCGACGCTGGCCG

GATCGGACGAACGAACGCGGCGCG GCTTTCTACCGGAGCGCTCCGCCGCATATC CACCGGCTACCCATTCGAAATGTCTCCCGA TGGCAATGTTGTGCGTATTTTTGGAATTTA TGGTGAGTTTGTCTATTTTGCGAGGGCGGT

ATATTTTTTATTGCGGGAACTGCCTCGGAT llePhePhe[leAlaGlyThrAlaSerAsD

GATAATCGGGATTCCGGGGAAAGTACAATT AspAsnArgAspSerGlyGluSerThrIle

GAAGGCCTGCAGAAAGCGCACATCGTCGGG GluGlyLeuGlnLysAlaHisIleValGly

i

ACCTTGTCGCTGGTCAATACAGCGTCCAGA ThrLeuSerLeuValAsnThrAlaSerArg

GATCAACGGTTGTTGAACAGGTCATTGTAT AspGlnArgLeuLeuAsnArgSerLeuTyr

GCATCCGGCAGCCAGTCTCAGCCGAGAGAG AlaSerGlySerGlnSerGlnProArgGlu

ATCAACGCGAAAACTCATGTCATATGGGCG IleAsnAlaLYsThrHlsValIleTrpAla

GCAAAATTTACATGTATTGAAGAATCAGGA AlaLysPheThrCysIleGluGluSerGly

AAGTCTTGATCGCCATTGCAATGACCACGG LysSer***

TCTGGGTCCTGCGAGACGTGGTCGGCCTGA GCGTCGCGGTGCGCTCGTGCGTACTGCCCG GCGAGAAGGTTCAGAAGGCGTGGCGCGAGC

Fig. 3. Nucleotide and deduced amino acid sequences of est from KWI-56. Nucleotides are numbered starting from the translational start site. Amino acids are numbered taking the first residue of the esterase. Asterisks indicate a stop codon.The underlined sequence is the consensus

sequence for the active site of serine enzyme.

81

HMSH-CF HMSH-PP HPDAH-PD TE-PP CE-KWI

1 MNAPQNSPEIGREIIAAGIRTNLHDSGAGFPLMMIHGSGPGVTAWANWRLVMPE 54 1 M N A P Q Q S P E I G R E I L A A G Y R T N L H D Q G E G F P A L L I H G S G P A - S P P G P T G A G S F R 53 1 MTALTESSTSKFLNIKEKGLSDFKIHYNEAGNGETVIMLHGGGPGAGGWSN---YYRN 55 I EIIPVPDQAAWNASKKSIQINDAIKMRYVEWGNPSGDPVLLLHGYTDTSRAFSS---LAPF 58 1 MIFHNGNVNLSYDVAGHGECIFFIAGTASDKSMWDG---LRQE 40

HMSH-CF HMSH-PD HPDAH-PD TE-PD CE-KWI

55 LAK .... SRRV I APDMLG- -FOYSERPADAQYNRDVWVDHAVGVLDALE I EQADLVGNISIFG 109 54 SSQ .... TR RV I APDMLG- -FGY SERPADGKYSQARWVEHA I GVLDALG I QQGD I VGNISlFG 108 56 I GPFVEAGYRVI LKDSPGFNKS-DAVVMDEQRGLVN-ARAVKGLMDALGI DRAHLVG~SNG 114 59 LSK .... DKRYLALDLRG--HG-GTSIPKCCYYVSDFAEDVSDFIDKMGLRNTTVIGHIS~G 112 41 LSG .... KYRTVAFDNRD--SG-EST I CDQPYTMLDLAKDALSVMDAEGLQKAH ] VGHL~LG 94

HMSH-GF HMSH-Pp HPDAH-Pp TE-Pp CE-KWI

I I0 GGIALALAIRHPERVRRLVLMGSAG-VSF-PIIEGLDAVWGYNPSFAEMRRL--LDIFAFD 166 109 GGLALALAIRHPERVRRLVLMGSVG-VSF-PITAGLETAWGYTPSLANMRRL--LDLFAHD 165 115 GATALNFAIEYPDRIGKLILMGPGGLGPSMFAPMPLEGIKLLFKLYAE-PSYENLKQMIQV 174 113 SMTAGVLASIHPDKVSRLVLISTA . . . . . . . LKTGPVLEWVYDTVLQK-DFP--LDDPSEF 163 95 GMIAQELAILAPDRVSTLSLVNTASRIDN-YMRSVIELARDWSKTITD-QRL--LNRSLYF 151

HMSH-CF HMSH-Pp HPDAH-Pp TE-Pp CE-KWl

167 RNLVNDELAELRYQASIRPGFHESFAAMFP-APRQRWVDGLAS-AEAAIRALPH-ETLVIH 224 166 RTLVNDELAELRYQASIRPGFQESFAAMFP-PPRQNGVDDLAS-NETDIRALPN-ETLVIH 223 175 FLYDQSLITEELLQGRWEAIQRQPEHLKNF-LISA-QKAPLSTWDVTARLGEIKAKTFITW 233 164 AKEWVAAPGKHDNGMAKNLKTEELAVPKHVWL-SAARGFSI-I-NWTAASKYLTAKTLILW 221 152 LALGSKALGSDIFNQVVDFASGSOSQPREA-LIRQ-WEIDLTV-DTTDRLSLINAKTHVIW 209

HMSH-CF 225 GREDQIIPLQTSLTLADWIARAQLHVFGQCGHWTQIEHAARFASLVGDFLAEADAAAIS 283 HMSH-Pp 224 GREDR I I PLQASLTLAQWl PNAQLHVFGQCC~HIWTQ I EHAERFARLVENFLAEADALHS 281 HPDAH-Pp 234 GRDDRFVPLDHGLKLLWN I DDARLHVFSKCGII-~WAQWEHADEFNRLA I DFLRQA 286 TE-Pp 222 GNQNQPMTESMQND I RAALPKAKF I QYNGFGIHISMFWEDPEMVAKDLNEFLK 272 CE-KWI 210 ASEDKI VTKDQQKMLVNGI SGAKFTCIEES~FPMI EAPEEFI RVLSGF I DKS 262

Fig. 4. Alignment of the predicted amino acid sequences of KW1-56 esterase and its homologous enzymes. Dashes indicate gaps introduced into the sequences so that the maximum homology may be obtained. The underlined sequence correspond to the consensus sequence of active serine observed in serine enzymes. Residues conserved in all the sequences are shown by asterisks. The boxed residues show the candidates constituting catalytic triads. HMSH-CF, HMS hydrolase from Pseudomonas CF600; HMSH-Pp, HMS hydrolase from P. putida; HPDAH-Pp, HPDA

hydrolase (HPDAH-Pp) from P. putida; TE-Pp, tropinesterase from P. putida; CE-KWI, esterase from KWI-56.

were treated with exonuclease III and mung bean nuclease to obtain unidirectional-deletion plasmids [13]. After single-stranded DNAs were isolated with the aid of helper phages (M13KO7), both strands were se- quenced over the whole region by dideoxynucleotide chain termination method [14] using the TFH sequence kit (Toyobo Co.).

The nucleotide sequence of a 1466 bp fragment between BamHI/Sau3AI and SalI is shown in Fig. 3. There was only one large open reading frame begin- ning with an ATG codon at nucleotide 1 and terminat- ing with a TGA codon at nucleotide 787. The molecu- lar weight calculated from the deduced amino acid sequence (262 amino acids) was 29011, which agreed well with the value estimated by SDS-PAGE (Fig. 2).

The amino acid sequence of KWI-56 esterase was compared with those of the other esterases from P. fluorescens [7] and B. stearothermophilus [6]. These three enzymes contained Gly-X-Ser-X-Gly, which is the consensus sequence for the active sites of lipase, esterase and serine proteinase [15-18]. However, there was no overall homology between them. Proteins ho- mologous to KWI-56 esterase were then searched for using EMBL and NBRF data base; 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid (HPDA) hydrolase [19] and tropinesterase [20], and 2-hydroxymuconic semialde- hyde (HMS) hydrolase [21] from P. putida, and HMSH from Pseudomonas CF600 [22] showed 28%, 25%, 30% and 26% homology to KWI-56 esterase, respectively.

Fig. 4 shows the alignment of the deduced amino acid sequences of these enzymes. They also contain the consensus sequence of active serine, their catalytic triads were believed to be composed of Ser, His and A s p / G l u by analogy with the serine hydrolases whose active site structures were well established [15-18]. On the basis of the conserved amino acids, Ser-92 and His-241 were tentatively assigned as the residues of the catalytic triad of KWI-56 esterase.

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