ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 296, No. 2, August 1, pp. 505-513, 1992
Purification, Molecular Cloning, and Expression of Lipase from Pseudomonas aeruginosa’ Mikiko Chihara-Siomi, Kazuhiro Yoshikawa, Yukihiro Sogabe,* Takuji Nakatani, Takaaki Institute for Chemical Research, Kyoto University, Toyobo Co., Ltd., Tsuruga, Fukui 914, Japan
Noriko Oshima-Hirayama, Kazumi Yamamoto,* Nishioka, and Jun’ichi Oda2
Uji, Kyoto 611, Japan; and *Tsuruga
Enzyme Plant,
Received December 20, 1991, and in revised form March 18, 1992
An extracellular lipase secreted by Pseudomonas aeruginosa TE3285 was purified. A genomic library of this strain was constructed in XEMBL3, and a DNA fragment 2.7 kb long containing the lipase gene, lipA, was isolated with an oligonucleotide probe synthesized on the basis of the partial amino acid sequence of a purified preparation of the enzyme. Nucleotide sequence analysis showed an open reading frame of 933 bases, and the deduced amino acid sequence agreed well with the molecular mass and partial amino acid sequences of mature lipase. The results of alignment of the amino acid sequences of five lipases from Pseudomonas species considered together with the published crystal structure studied with human pancreatic lipase showed that Ser82, His251, and Asp209 were catalytic residues and that a surface loop from residues 172 to 204 was responsible for the substrate specificity. About 50 bases downstream of lipA, there was another gene, 1ipB. The sequence of ZipB was highly homologous to that of putative modulators of the production of active lipases in other Pseudomonas species. Expression plasmids encoding 1ipA followed by the complete or incomplete 1ipB gene downstream of the Zac promoter of pUC18 were constructed. 1ipA was expressed in Escherichia coli 1100 o 1992 only in the presence of the complete 1ipB gene. Academic Press, Inc.
Lipases (EC 3.1.1.3) are used for organic synthesis even in nonaqueous solvents (l-4). By using lipase from Pseudomonas species in anhydrous organic solvents, we have brought about the kinetic resolution of racemic halohydrins (5), hydroperoxides (6), and cyanohydrins (7-9) and 1 Part of this work was presented at the CEC-GBF International Workshop, Braunschweig, West Germany, September 1990. ’ To whom correspondence should be addressed. 000%9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
the asymmetric ring opening of cyclic anhydrides (10,ll). Our attempts to resolve [ l,l’-binaphthyl] -2,2’-diol and its acylated derivatives succeeded only with a lipase from Pseudomonas aeruginosa TE3285, isolated from soil in Tsuruga, Japan (12). In addition to this substrate specificity, this enzyme efficiently processes triglycerides into free fatty acids and glycerols and improves the stability when it is immobilized. To understand these properties, analysis of the molecular structure of the enzyme is essential. Amino acid sequences of lipases from several Pseudomonas species have been deduced from the nucleotide sequence of their genes (13-18), but all lipases with known crystal structures are of eukaryotic origin (19-22). Some lipase genes from Pseudomonas species have another gene downstream (13-16). The protein from this gene has not been isolated or characterized yet, but is responsible for the production of active lipase. P. aeruginosa TE3285 secretes a lipase extracellularly, but the lipase gene has not been isolated. In this study, to clarify the structure-function relationship of this enzyme, molecular cloning of the lipase gene, lipA, was done. The amino acid sequence of the enzyme deduced from the nucleotide sequence was compared to the sequences of other lipases registered in databases. A nucleotide sequence downstream of 1ipA was later identified as being another gene, 1ipB. Expression of 1ipA in Escherichia coli was tested in the presence and absence of 1ipB. MATERIALS
AND
METHODS
Bacterial Strains and Plasmids The source of chromosomal DNA for cloning was l? aeruginosa TE3285, isolated from soil in Tsuruga, Japan. E. coli P2392 [PP lysogen of LE392 [F-, hsdR514, supE44, supF58, lacy1 or A(iucZY)6, galK2, gaZT22, metB1, trpR55, X-, ncrA-, mcrB+]] (23) (Stratagene, La Jolla, CA) was used for construction of the genomic library and as host cells of recombinant phages. E. coli JM109 [mAI, e&Al, gyrA96, thi, hsdRl7, supE44, relA1, X-, A(Zac-proA@, [F’, proA& lucPZAM15, traD36]] (24) 505
506
CHIHARA-SIOMI
(Takara Shuzo Co., Ltd., Kyoto, Japan) was used for subcloning in plasmids. E. coli 1100 [F-, prototrophic, endo Z-1 (25) was used for expression of the lipase gene. XEMBLB (26) (Stratagene) was used for cloning, and pUC19 (Takara Shuzo) and M13mplS (Takara Shuzo) (24) were used for subcloning and sequencing. pUCl8 (Takara Shuzo) (24) was used to construct expression vectors for the lipase gene. Transformation was done by the method of Hanahan (27).
Lipase Assay The protein concentration was assayed with Coomassie protein assay reagent (28) (Pierce, Rockford, IL). Lipase activity was measured with a Lipase Kit S (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) at 30°C. In this assay method, thiol groups liberated by the hydrolyzation of 2,3-dimercaptopropan-l-01 tributyrate are coupled with 5,5’-dithiobis(2-nitrobenzoic acid), and the absorbance of the resulting B-thio2-nitrobenzoate anion at 412 nm is measured (29).
Purification
Japan) with 0.1% (v/v) trifluoroacetate and a linear gradient of O-60% (v/v) acetonitrile. 3. Digestion with lysylendopeptidase (Achromobacter proteinase Z). Lipase (0.46 mg) was denatured overnight in 20 mM Tris-HCl (pH 8) containing 7 M urea, diluted to 3.5 M urea, and digested with 1.65 pg of lysylendopeptidase (Wako Pure Chemical Industries, Osaka, Japan) at a 1:400 (w/w) ratio of peptidase to lipase for 9 h at 30°C. The digest was fractionated as described above. 4. Cleavage with cyarwgen bromide. Lipase (3 mg) was treated with 1000 equivalents of CNBr in 70% formic acid for 24 h at 25°C. The hydrolysate was lyophilized. Peptide fragments were separated on 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane in 10 mM 3-(cyclohexylamino)1-propanesulfonate buffer (pH 11) containing 10% ethanol (30).
Sequencing of N-Terminal
Amino Acids
The amino acids at the N-terminal of the lipase and of the peptide fragments obtained above were sequenced with a gas phase sequencer (Applied Biosystems Model 470A).
of Lipase
P. aerugginosa TE3285 was grown at 30°C for 30 h in 6 liters of culture medium (pH 6.5) containing 20 g of polypeptone, 4 g of yeast extract, 4 g of meat extract, 2 g of KHzPO,, 0.5 g of MgS0.e 7Hz0,0.5 g of KCl, 5 g of purified rice bran oil, and 1 g of silicone per liter. The culture medium was centrifuged and the supernatant was brought to 35% saturation with (NH&SOI. The mixture was centrifuged and the pellet was suspended in 200 ml of 50 mM potassium phosphate buffer, pH 7.5, containing 2 mM MgClr and 0.5 mM EDTA. The solution was put on a column of octyl-Sepharose CL-GB, which was washed, and equilibrated with 20 mM phosphate buffer (pH 7.5) containing 2 mM MgCl, and 0.5 mM EDTA. Fractions were eluted with the same buffer containing 1% Triton X-100. Active fractions were collected and further fractionated on DEAE-Sepharose with a gradient of NaCl (O-l.0 M). The fraction containing lipase activity was pooled, precipitated by the addition of (NH,)zSOI to 60% saturation, and recovered by centrifugation. The pellet was suspended, put on a Sephadex G-25 column, and desalted. The active fractions were collected and lyophilized.
Amino Acid Analysis A purified preparation of lipase (50-100 /.rg) was reduced with 1.3% (v/v) mercaptoethanol, carboxymethylated with 1.44 M iodoacetate, and hydrolyzed with 6 N HCl at 110°C for 22, 48, or 72 h. Cysteine and cystine were analyzed as cysteic acid after oxidation of lipase (100 pg) with performic acid. The hydrolysates were analyzed with an amino acid analyzer (Hitachi Model 835).
Chemical and Enzymatic Preparation of Lipase
ET AL.
Digestion of a Purified
1. C-terminal sequence. Lipase (1.4 mg) was dissolved in 0.1 M phosphate buffer (pH 8.75) containing 0.8% SDS3 and digested with 35 pg of carboxypeptidase A-DFP (Sigma, St. Louis, MO) at a 1:40 (w/w) ratio of peptidase to lipase for 10 to 120 min at 25°C. 2. Digestion with t~ypsin. Lipase (3 mg) was reduced, carboxymethylated, denatured in 8 M urea overnight, and digested with 50 pg of trypsin-TPCK (Sigma) at a 1:50 (w/w) ratio of trypsin to lipase in 0.1 M Tris-HCl (pH 8) containing 2 M urea for 36 h at 37°C. The digest was fractionated on an Ultron N-&s column (Shinwa Kako, Kyoto,
3 Abbreviations used: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TPCK, ZV-tosyl-L-phenylalanyl chloromethyl ketone; pfu, plaque forming units; SSCP, a mixture of sodium chloride, sodium citrate, and phosphate; IPTG, isopropyl-fl-D-thiogalactopyranoside; DFP, diisopropyl fluorophosphate.
Construction
of a P. aeruginosa Library
Chromosomal DNA of P. aeruginosa TE3285 was isolated from 500ml culture medium and partially digested with MboI. After agarose gel electrophoresis, DNA fragments 7 to 23 kb long were isolated on a DEAE membrane (Schleicher and Schuell, Dassel, Germany), ligated into BamHI-cut arms of AEMBLS (Toyobo, Osaka, Japan), and packaged with commercially available packaging extract (Gigapack Gold, Stratagene).
Screening of the Library for the Lipase Gene E. coli P2392 cells infected by recombinant phages were plated on agar at a density of 10’ pfu/S2-mm dish. Plaques were transferred to nitrocellulose filters. The DNA was denatured first with 0.5 M NaOH containing 1.5 M NaCl and then with 0.5 M Tris-HCl, pH 7.5, containing 1.5 M NaCl. The filters were baked at 8O’C for at least 2 h under reduced pressure. Prehybridization was done in 5 X SSCP (1 X SSCP contains 6 mM NaCl, 0.75 mM Na-citrate, 0.65 mM KHzPO,, and 0.05 mM EDTA), 5 X Denhardt’s reagent [l X Denhardt’s contains 0.02% bovine serum albumin, 0.02% Ficoll 400 (Pharmacia, Uppsala, Sweden), and 0.02% polyvinylpyrrolidone], 0.5% SDS, and 0.02 mg/ml salmon sperm at 65°C for 1 h. The filters were hybridized with the oligonucleotide probe (lo7 cpm per filter) in 5 X SSCP, 5 X Denhardt’s reagent, and 0.5% SDS at 50°C for 16 h, and washed in 6 X SSCP first for 5 min at room temperature and then for 20 min at 42’C. Positive clones were isolated.
Nucleotide
Sequencing
DNA sequencing was done by the chain termination method with T4 DNA polymerase (Sequenase DNA sequencing kit, United States Biochemicals, Cleveland, OH) or Thermus thermophilus DNA polymerase
g41k t7kf3k 3;k
20 k 1
14.4 k 1
1 2 FIG. 1. SDS-PAGE of purified lipase from Pseudomonas aeruginosa TE3285. Lanes: 1, molecular mass standards, values in daltons; 2, purified lipase.
CLONING
AND EXPRESSION
P. aerugimsaTE3285DNA(ca.13kb)
OF PSEUDOMONAS
507
LIPASE
'
lII/BamHI)
KpK
~uC19 pUL1
4s=>i::,,
r------
439
'",:I
Sal1 I !-Q-IS
844 I
1087 PscI
1ipA c
2007 2117 P.q.3 Pyu1
lipB
-
-
c
*
-
*
-
-_____t t
b -
-
t
c c
w -
-
*
--
-
+ -
I
t
--
--
t --
1199 AqcI
-
e
-
4
4 c L
--
FIG. 2. Partial restriction map of pUL1 containing the lipase gene from P. aeruginosa TE3285, and sequencing strategy. Nucleotide numbers of sites cleaved by selected restriction endonucleases are given. Arrows in the bottom open box indicate open reading frames. The XEMBLB derivative obtained from positive clones of E. coli P2392 screened with an oligonucleotide probe based on the complement of the amino acid sequence of mature lipaseis also shown.
(TTH DNA sequencing kit, Version 2.0, Toyobo). The synthetic oligonucleotides used as the probe for cloning were also used as a primer for sequencing.
Lip&e Assay Expressed in E. coli Lipolytic activity of the E. coli transformants was tested on tributyrin plates containing 0.25 X LB (1 X LB contains 1% Bacto tryptone, 0.5% Bacto yeast extract, and 1% NaCl), 0.4 mM IPTG, 1% tributyrin, and 1.5% (w/w) agar. Cells were incubated on the tributyrin plates first for 24 h at 37°C and then for 24 h at 4°C. In liquid culture, induction of
TABLE Amino
I
Acid Composition of Mature from P. aeruginosa TE3285
Lipase
Amino acid
Amino acid analysis0
DNA sequence
Ala CYS
23.1 2.4 28.4 24.1 9.6 32.4 7.3 10.5 7.0 29.1 2.9 12.6 10.0 36.0 18.0 20.2 9.5
23 2 27 22 10 31 8 11 7 30 3 13 11 35 17 22 2 11
Asx Glx Phe GUY His Ile LYS Leu Met Pro Arg Ser Thr Val Trp Tyr
’ Calculated from the total of 283 amino acid residues. Two Trp residues were missing from the 285 deduced from the nucleotide sequence.
the lot promoter with 1 mM IPTG was done after 2.25 h of incubation. After 12 h of incubation in 4 or 40 ml of culture medium with vigorous shaking at 37”C, a crude cell extract was obtained by ultrasonication with a Sonifier 250 (with microtip, output of 2, duty of 50%; Branson, Danbury, CT) in 1 or 2 ml of 20 mM Tris-HCl (pH 8.0) containing 200 mM NaCI, respectively. Lipase activity in the crude extract was measured as described above. RESULTS
Purification
of Lipase
A lyophilized sample of 193 mg with lipase activity of 1.6 X lo5 pmol/min per milligram was obtained. SDSPAGE of the purified lipase is shown in Fig. 1. The purity of the sample was about 95%. This preparation was analyzed for its amino acid composition and partial sequences. Oligonucleotide
Probe for Screening of the DNA Library
The first 25 residues on the N-terminal were Ser-ThrTyr-Thr-Gln-Thr-Lys-Tyr-Pro-Ile-Val-Leu-Ala-HisGly-Met-Leu-Gly-Phe-Asp-Asn-Ile-Leu-Gly-Val-. One of the peptide fragments isolated from the digest cleaved with CNBr had Leu-Gly-Phe-Asp-Asn-Ile-LeuGly-Val-Asp-Tyrat its N-terminus, which completely matched the sequence following Met16 in the N-terminal sequence of the lipase. A DNA probe for screening of the genomic library was designed based on the complement of the sequence, Met-Leu-Gly-Phe-Asp-Asn-Ile, and a mixture of eight oligonucleotides (21-mers) with degeneration at three sites, 5’-ATGCTGGGCTT(C/T)GA(C/ T)AA(C/T)ATC-S, was synthesized. The 5’-end of the probe was labeled with 32P by use of [-y-32P]ATP (Amersham, Buckinghamshire, UK) and T4 polynucleotide kinase (Takara Shuzo) .
508
CHIHARA-SIOMI
ET AL.
GTCGACCATTTCAGCCTGTTTTGCTCGCAARACGACGCCGCGGGCGTGCGCACCG~CAC TCGGTCGCTGGGCGTTGTGCGGGWVLWLTTCAAACGACCGTTTCGCGCCGTMCMCCCG rrTCTTCCGCTCTGCCACGCAGGTTATGACCGGCCGCCAGGMGCCGCG~TTTCCTGGC CTGGAGGAAAAAA GCCGMGCTGGCACGGTTCCTGCGCAAGGGACAGCGAAGCGGTTCTC
1383; CGGCGCCCCCGGCCAGCCCGCAGGCGGGCGCAGACCGCGCCCCGCCAGCAGCCTCC~GG APPASPQAGADRAPPAASAG '""5; GAGAAGCGGTGCCGGCCCCCCAGGTCATGCCGGCCMGGTCGCGCCGCTGCCMCCTCCT EAVPAPQVMPAKVAPLPTSF 1507: TCAGGGGCACCAGCGTCGATGGCAGTTTULGTGTGTCGACGCCAGCGGC~CTGCTGATCA RGTSVDGSFSVDASGNLLIT
CCGGMGGATTCGGGCWLTGGCTGGCAGGACGCGCCCCTCGGCCCCATC~CC~G
1561 CCCGCGACATCCGCMCCTGTTCGACTACTTCCTCAGCGAC 91 RDIRNLFDYFLSDGEEPLQQ
AGACAACATWLAGM~TCTCTGCTCCCCCTCGGCCTGGCCATCGGCCTCGCCTCTCT MKKKSLLPLGLAIGLASL
421 GGCCCACGGCRTGCTCGGCTTC~~CATCCTCGGGGTCGACTACTGGTTCGGCATTCC 13 A H G&j 7. R C 11111 lb. G V D Y W F G 1 P 481 CAGCGCCTTGCGCCGTGACGGTGCCCAGGTCTACGTCACCGMGTCAGC~GTTGGACAC 33 S A LRRD GAQ VY VT E V S Q LD T 541 CTCGGMGTCCGCGGCGAGCTTGCTGCAACAGGTGGAGG~TCGTCGCCCTCAGCGG 53 S E VRE E 0 L L 0 0 VE E I VA L S G 601 CCAGCCCAAGGTCAACCATCGGCCACAGCCACGGCGGGGTCGC 730 P KVN L I G H S H GG P T I RYVA
1;:;
AAAGCCTGGACGGCCTGCGCGCCTACATCGCCGCCGAACTCC SLDGLRAYIAAELQEPARGQ
'I":;
AGGCGTTGGCGCTGATGCAGCAATACATCGACATCGAA ALALMQQYIDYXKELVLLER
1;;;
GCGACCTGCCGCGCCTGGCCGACCTCWLCGCCCTGCGCCA DLPRLADLDALRQREAAVKA
1;;;
CCCTGCGCGCGCGGATCTTCAGCMCGMGCGCACGTGGCGTTCTTCGCCGACGAG~ LRARIFSNEAHVAFFADEET
1;;;
CCTACMCCAGTTCACCCTGGAGCGCGCCTGGCCACG YNQFTLERLAIRQDGXLSTE
1;:; 661 CGCCGTACGTCCCGACCn;ATCGCTTCCGCCACCAGCGTCGGCGCCCCG~CMG~GT~C 93AVRPDLIASAe 1;:; 721 W;ACACCGCCGACTTCCTGCGCCAWLTCCCCACCGGGTTCGGCCGGC~GGCMTCCTCTC llS_IL_T_ADFLRQXPPGSAGEAILS 2;;; 781 CGGGCTGGTCAACAGCCTCGCGCGCTGATCAGCTTCCTTTCCAGC~C~CACCGGTAC 133G LVN S LGAL I S FL S S G S T G T 2:;; 841 GCAGAATTCACTGGGCTCGCTGGAGTCGCTGAACAGCGAGGGGGCC~GCGCTTC~CGC 153 QNS L G S LES LN SE GAARFNA 2;;; 901 CAAGTACCCGCATGGCGTCCCCACCTCGGCCTGCGGCGAGGGCGCCTAC~GGTC~CGG 173 K r P H G VPT S AC GE G AYKVNG 2::; 961 CGTGAGCTATTACTCCTGGAGCGGTTCCTCGCCGCTGACCMCTTCCTC~TCCGAGCGA 193vS Y Y ,5 W S G S S P T. T N F T. II P S D 2:;; 1;;; CGCCTTCCTCGGCGCCTCGTCGCTGACCTTCMG~~C~CC~CC~C~C~GC~TG~T AFLGASSLTFK 2341 1081 CGGCACCTGCAGTTCGCACCTGGGCATGGTGGTGATCCGCGACMCTACCGGATGMCCACCT 2401 233GTCSSHLG,MVIRDNYRMNHL
AGGAAAAGGCCGCCGCCATCGACCGCCGCCTGCGCGCCAGCCTGCCGG~GACCAGCAG~ EKAAAIDRLRASLPEDQQES GCGn;CTGCCGCMCTGCACGMCTGCAGCAGCAGACCGCCGCCCTCCAGGCC~TG VLPQLQSELQQQTAALQAAG GCGCCGGCCCGGMGCCATCCGCCAGATGCGTCAGCMCTGGTGGGCGCCGMGCC~CA AGPEAIRQMRQQLVGAEATT CCCGCCTGGAGCMCTCGATCGGCMCGCTC~CCTGGMGGGCCGGCTG~CGACTATT RLEQLDRQRSAWKGRLDDYF TCGCCGAGAAGAGCCGWLTCGMGG~TGCCGGGCTGAGCGMGCCGACCGCCGC~GG AEKSRIEGNAGLSEADRRAA CGGTCGMCGCCTGGCCGAGGAGCGCTTCAGWLGC VERLAEERFSEQERLRLGAL TGGAACAGATGCGCCAGGCCGAGCAGCGCTGACCGGCACGGAAA~GAACG~ EQMRQAEQR MGGGCGCTTCGGCGGATMCGCTACCCTCAGGGGTGCAGGG CGGAAACCTGTGCTGCGCGCCGCMCG~GGGCGGCCCCG~GGTGTCCGCCCTT
2461 TTTCGTCGCCAGCCCGGTTCAGCGGWLCAGCTTGCCGTCCAGCGAGMCTTGCCGGCGCC
1141 GGACGAGGTWULCCAGGTCTTCGGCCTCACU\GCCTGTTCG~AC~GCCCG~TCAGC~T 253 D E VN Q VF G L T S LF E T S P S
2521 ATCGRTCAGCRGCGCW\CGCTGATCATCATCAGCAGTCAGGGCATATTCATAGCCGTTGTC
1201 CTACCGCCAGCACGCCMCCGCCTGAAGMCGCCAGCCTGTAGGACCCC&U@X&&T 273 Y R Q H A N R L K N &-J.-JI-. CTTTCCC&&CCCCTCGCGTGAAGAAMTCCTCCTGCTGATTCCAC MKXILLLIPL 12Y c-
2581 GGTWLTGAAGAAGCCATTGCCGATGTGCACGCTGAAGATCGCCAC~TCCGTTGACCTGC 2641 AGGTCGACCCAGATC
TGGCGTTCGCCGCCAGCCTGGCCTGGTTCGTCTGGCTG~CCTTCCCCCGCCCCCGAGA AFAASLAWFVWLEPSPAPET
FIG. 3. Nucleotide sequence and the deduced amino acid sequence of the 2.7-kb region containing 1ipA and 1ipB genes from P. aeruginosa TE3285. The putative Shine-Dalgarno sequences are boxed. The vertical arrow indicates the position of the release of the signal peptide, deduced from the N-terminal amino acid sequence of the purified preparation of the enzyme. The underlined peptide sequences deduced from the sequence of 1ipA were found independently by trypsin or lysylendopeptidase digestion and by cyanogen bromide cleavage, except that the serine residue at position 71 was not detected by the amino acid sequencer. The C-terminal sequence with the dashed line was found by carboxypeptidase A analysis. The amino acid sequence used for the oligonucleotide probe for screening is boxed. The amino acid sequence deduced from the sequence of 1ipB is in italics.
Isolation of the P. aeruginosa Lipase Gene Some 9 X lo5 packaged recombinant phages were obtained with 0.2 pg of DNA fragments. The calculated redundancy of the library was about 3 X 103, assuming that the genome size of the strain was 4 X lo6 bases. Nine positive plaques out of 4 X lo5 plaques were selected. DNA from the positive clones was digested with the restriction endonucleases SalI, KpnI, BgZII, and P&I and studied by Southern blotting. The results suggested that a 4.3-kb KpnI-BglII fragment contained the lipase gene. This fragment was subcloned into pUC19 vector, giving pUL1. Nucleotide Sequence Analysis The nucleotide sequence4 of a region of 2.7 kb was identified. A partial restriction map and the sequencing 4 The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the accession number of D10048.
strategy for the region are shown in Fig. 2. An open reading frame starting from nucleotide 308 coded for a polypeptide of 311 amino acid residues (Fig. 3). The G + C content of the 2.7-kb region was 66.9%, which reflected the high G + C content (67.2%) of the P. aeruginosa genome (31). The G + C contents of the first, second, and third positions of the codon triplets for this open reading frame were 59.8, 46.9, and 91.0%, respectively. In general, genes from Pseudomonas have a high G + C content in the wobble base position (32,33), which helps in the checking of the location and accuracy of the protein-coding sequence. The deduced amino acid sequence from the DNA sequence completely matched the 116 residues obtained by the analysis of the N-terminal amino acid sequence of the lipase and the peptide fragments generated by endopeptidase digestion or cyanogen bromide cleavage. The three residues at the C-terminal matched those found by digestion with carboxypeptidase A. Therefore 1ipA was the lipase gene.
CLONING
AND
a) P. P. P. P. P.
EXPRESSION
0
OF PSEUDOMONAS
l
-QLDTS-E FQSDDGPN FQSDDGPN FQSDDGAN -AANDN-E
aeruginosa TE3285 cepacia ~-12-33 cepacia DSM 3959 sp. KWI-56 fragi IFO-12049
GSAGEAILSGLVNSLGALIS YDPT-GLSSTVIAAFVNVFG YDPT-GLSSSVIAAPVNVFG YDPT-GLSSSVIAAFVNVFG GRLGETVAAALTTSFSAFLS
TE3285 ~-12-33 DSM 3959 KWI-56 IFO-12049 -PTSACGEGA-YK GAPGSCQTGAPTE GAPGSCQTGAPTE GAPGSCQTGAPTE -PDRWGGMGP-AQ
TE3285 M-12-33 DSM 3959 KWI-56
IFO-12049 ~'33285 M-12-33 DSM 3959 KWI-56 IFO-12049
509
LIPASE
SDAFLGASSLTF--KNGT STLALFGTGTVMVNRGSG STLALFGTGTVMINRGSG STLALFGTGTVMINRGSG LHNALRVFDSFFT-RETR
SG ss ss SS GH
---------------------SSPL AIQPTISVFGVTGATDTSTIPLVDP AIQPTLSVFGVTGATDTSTLPLVDP AIQPTLSVFGITGATDTSTVF'LVDL IIK---------------GSRL~S VFGLTSLFETSPVSVYRQHANRLKNASL LLGVRGANAEDPVAVIRTHANRLKLAGV LLGVRGAYAEDPVAVIRTHANRLKLAGV LLGVRGAYAJXDPVAVIRTHANRLKLAGV MARGSAGASTR
148 152 152
152 150
204 237 237
237
212 285 320 320 320
277
b) KWI-56 DSM 3959 TE3285 168 168 162
257 251
251
FIG. 4. (a) Comparison of the amino acid sequences deduced from the nucleotide sequences of five lipases from Pseudomonas species. Identical residues are boxed. The serine residue in a conserved sequence pattern among lipases and serine proteases is marked with an asterisk. Open and closed circles and the closed inverted triangle indicate the conserved histidine, aspartic acid, and tryptophan residues,, respectively. Numbers refer to amino acid positions. Gaps indicated by dashed lines were introduced to give maximum similarity. (b) Comparison of predicted amino acid sequence of ZipB with two other putative modulator or activator genes from Pseudomonas species necessary for the expression of lipase activity.
The amino acid sequence of the purified preparation of the Iipase began at Serl, so the 26 residues from the initiator methionine to Ala were predicted to be the signal sequence. von Heijne (34) suggested that signal sequences consist of positively charged residues near the N-terminus, followed by a long stretch of hydrophobic amino acids and ending with an amino acid with a small side chain. The putative signal sequence of this lipase matched this description: there were three positively charged lysine residues from Lys(-25) to Lys(-23), a hydrophobic core consisting of 19 residues from Leu(-21) to Ile(-3), and Ala at the end. The molecular mass of the mature protein of 285 residues was calculated to be 30,145 Da. This was in agreement with the apparent molecular mass of 28 X lo3 Da estimated on SDS-PAGE (Fig. 1). The amino acid composition calculated showed good correspondence with that
found in the purified preparation of the lipase (Table I). The content of serine residues in the lipaae was high (12%) compared with the mean serine content, 5.5%, of the 107 proteins from P. aeruginosa registered in the NBRF database (release 30.0). Figure 4a shows the alignment of the amino acid sequences of five lipases from Pseudomonas species. The amino acid sequence of TE3285 lipase was identical at 38.1,36.6, 38.1, and 41.8% of its positions to lipases from M-12-33 (15), DSM 3959 (13), KWI-56 (16), and IFO12049 (18), respectively. Another gene, the initiation codon of which was GTG, was found 50 bases downstream of the stop codon of ZipA and was named ZipB. The G + C content of ZipB (75.2, 45.7, and 86.1% in the first, second, and third positions of codon triplets, respectively) was typical of genes from Pseudomonas species (32, 33). For Pseudomonas cepacia
510
CHIHARA-SIOMI
ET AL.
from 40 ml of culture medium incubated for 12 h at 37°C in LB broth was 8.9 X 10’ bmol/min per milligram. These results showed again that 1ipA was the lipase gene. TO test whether 1ipB was required for the expression of lipase activity, three deletion plasmids were constructed (Fig. 5). The DNA fragment from nucleotides 2348 to Ecd)lDp1 Yes NO 2655 in Fig. 3 was deleted from pULl0 with exonuclease FIG. 6. Construction of expression vectors for the P. aeruginosa III and mung bean nuclease (Toyobo) to give pUL11, TE3285 lipase gene. An NcoI restriction site was introduced in each which contained both 1ipA and 1ipB. Eco47111or EcoO1091 vector at the initiation codon of 1ipA by site-directed mutagenesis with a synthetic oligonucleotide. pULl0 was digested with Eco47III or digestion gave two deletion plasmids, one that lacked a EcoO1091 and fragments of 63 bases and 1.8 kb were released, giving part of 1ipB (pUL12) and one that lacked all of 1ipB pUL12 and pUL13, respectively (one of the EcoO1091 restriction sites (pUL13). E. coli llOO/pULll cells had lipolytic activity, of pULl0 originated from pUC18). but llOO/pUL12 and llOO/pUL13 cells did not. After culture of these transformants in 2 ml of LB broth, the lipase activity in a crude extract was assayed and found to be M-12-33, DSM 3959, and Pseudomonas sp. KWI-56, the 1.0 X 103,9.3 X lo’, 8.5 X lo-‘, and 9.6 X 10-l pmol/min expression of their lipase genes in E. coli depends on the per milligram for E. coli 1100 cells transformed with coexistence of another gene immediately downstream pUL10, pUL11, pUL12, and pUL13, respectively. from the lipase gene (13-16). The nucleotide sequence and deduced amino acid sequence of 1ipB were compared DISCUSSION with those of DSM 3959 and KWI-56. 1ipB was 52-54% Lipases and lipoprotein lipases have a sequencepattern, identical in its overall nucleotide sequence and 27% idenGly-X1-Ser-X,-Gly (where X is any amino acid), similar tical in the deduced amino acid sequence (Fig. 4b). Flanking regions of 1ipA and 1ipB were analyzed. The to one common to serine protease families. Studies of the transcriptional start site for lipA was searched for up- three-dimensional structure of lipases from human pancreas (19), Rhizomucor miehei (20), and Geotrichum canstream of the initiation codon of lipA. In P. aeruginosa, didum (21) showed that a catalytic triad resembling that sequences AAAAACCACCCG and AAAAACCAACCG were found near the transcriptional start of the hemolysin of serine protease and consisting of serine, histidine, and (35) and exotoxin A (36) genes, respectively. A similar aspartic or glutamic acid residues is present in the strucsequence, AAAAACACCCTG, was found upstream of the ture of these lipases. The serine residue of the catalytic Pseudomonas fragi IFO-12049 lipase gene. No such se- triad was the same one that is part of the shared sequence pattern. In rat hepatic lipase, site-directed mutagenesis quence was found upstream of 1ipA or 1ipB. The putative ribosome binding sequences of the two genes were as- of the serine residue to glycine in the shared sequence signed to nucleotides 295-298 and 1280-1283, respec- pattern resulted in the complete loss of both lipolytic and esterase activities (38), suggesting the importance of this tively. Downstream of these genes, there were palindromic sequencesbetween nucleotides 1250 and 1271 and between serine residue for catalysis. P. aeruginosa TE3285 lipase contained a sequence, Gly-His-Ser82-His-Gly, in which 2325 and 2340; these might be p-independent termination Ser82 seemed to be the active residue in the catalytic signals. triad as seen in other lipases. Table II compares the sequence from six residues upstream to two residues downExpression of 1ipA in E. coli 1100 stream of Ser82 in P. aeruginosa TE3285 lipase with the To check further the identity of the gene product of same sequence in lipases and lipoprotein lipases from both lipA, we set out to express 1ipA in E. coli (Fig. 5). In this prokaryotes and eukaryotes. In P. aeruginosa TE3285 liexperiment, an NcoI restriction site was introduced in pase, X, was histidine, but in the other enzymes, it the region of the initiation codon of 1ipA by site-directed was not. The positions of the catalytic histidine and aspartic mutagenesis by the method of Kunkel (37) with a synthetic oligonculeotide corresponding to the sequence from acid residues of P. aeruginosa TE3285 lipase could be proposed by sequence alignment with the other four Pseunucleotides 298 to 321: 5’-ATGAGACACCATGGAGAAGAAGTC-3’. (The mismatched bases at 306 and 311 are domonas lipases because functionally important residues underlined.) The DNA fragment from nucleotides 229 to are often conserved in the sequences of functionally re2655 was inserted downstream of the pUC18 lac promoter lated enzymes. Among the five sequences, three histidine (SmaI cloning site), giving pUL10. E. coli 1100 cells and five aspartic acid residues were conserved. From setransformed with pULl0 (llOO/pULlO) were cultured on quence similarities of the region with portions of the litributyrin plates and lipolysis was seen as a clear zone pases and lipoprotein lipases from mammals (Table III), formed around colonies because of degradation of the lipid His251 and Asp209 of P. aeruginosa TE3285 lipase seemed emulsion. The lipase activity in the crude extract prepared to be catalytic residues. This assignment was further
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LIPASE
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Conserved Amino Acid Sequence in Lipases and Lipoprotein Lipases from Various Sources Reference”
Sequence
Organism
Protein
P. aeruginosa TE3285 Pseudomonas cepaciu DSM 3959 Pseudomonas cepacia M-12-33 Pseudomonus sp. KWI-56 Pseudomonas fragi IFO-3458 Pseudomonns fragi IFO-12049 Pseudomonas fltwrescens SIK WI Staphylococcus aweus Staphylococcus hyicus Morarella sp. Morarella sp. Candida cylindracea Geotrichum candidum Geotrichum candidum Human milk Rat Human Rat Penicillium camembertii Rhizomucor miehei Humicola lamugirwsa Pig Human Dog Mouse Rabbit Rat Mouse Human Chicken Guinea pig Mouse cow Human
Lipase Lipase Lipase Lipase Lipase Lipase Lipase Lipase Lipase Lipase 1 Lipase 3 Lipase Lipase I Lipase II Bile salt-activated lipase Adipose tissue hormone sensitive lipase Gastric lipase Lingual lipase MDG lipase’ Lipase Lipase Pancreatic lipase Pancreatic lipase Pancreatic lipase Pancreatic lipase Hepatic lipase Hepatic lipase Hepatic lipase Hepatic lipase LPLd LPL LPL LPL LPL
76* VNLIGHSHG VNLVGHSQG VNLVGHSQG VNLVGHSQG VNLIGHSQG VNLIGHSQG VLVSGHSLG VHLVGHSMG VHFIGHSMG L GAIGWSMG T HVGGNSMG VMIFGESAG VMIFGESAG VMIFGESAG TLFGESAG I CLAGDSAG I LHYVGHSQG HYVGHSQG I LVVVGHSLG V AVTGHSLG VVFTGHSLG VHVIGHSLG VHVIGHSLG VQLIGHSLG VHLIGHSLG VHLIGYSLG VHLIGYSLG VHLIGYSLG VHLIGYSLG VHLLGYSLG VHLLGYSLG VHLLGYSLG VHLLGYSLG VHLLGYSLG
84 T 13 14 16, N N 18, N 17 N N N N 42 N N N N N N 43 N 44 N N N N 45 N 46 N N N 47 N N
’ T, this study; N, registered in NBRF database (release 30.0). * The numbers indicate the amino acid positions for P. aeruginosa TE3285 lipase. ’ MDG lipase, mono- and diacylglycerol lipase. ’ LPL, lipoprotein lipase.
tested by comparison of the secondary structures of the lipases predicted by the joint prediction method of Konishi and Nishikawa (39). By the application of this method to human pancreatic lipase, the secondary structure around the catalytic histidine and aspartic acid residues was predicted to be an a-helix at His‘263 and a coil at Asp176. These assignments were consistent with those observed by crystallographic analysis, indicating the reliability of the method. His251 and Asp209 of P. aeruginosa TE3285 lipase were predicted to be an a-helix and a coil, respectively. Both residues were part of the same secondary structure as the catalytic histidine and aspartic acid residues of human pancreatic lipase, so this identification of the catalytic residues is reasonable. For plans for the protein engineering of P. aeruginosa TE3285 lipase, identification of the sequence region that
governs the reaction specificity of the enzyme would be helpful. Crystallographic analysis of lipases (19-21) showed that they have a surface loop called a “lid.” The loop seemed to move from the catalytic site, possibly through interfacial activation of micelles, leaving the site accessible to its substrate. In P. aeruginma TE3285 lipase, a surface loop was suggested from the following results. First, the joint prediction method predicted that residues 172-204 form a random coil except for residues 195 and 196, predicted to be a short @-strand. This coil, composed of 33 amino acid residues, was the longest in this lipase, and the length of the loop corresponded roughly to that of human pancreatic lipase (25 residues). Second, upon tryptic digestion of P. aerugimsa TE3285 lipase, a peptide fragment produced by cleavage at Tyr196 was obtained at high yield in the early stage of digestion. Considering
512
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ET AL.
TE3285 was effective for the lipase-catalyzed reaction of a bulky substrate such as binaphthol might be due to this structural difference around residue Trp198. Expression studies of pUL10, pUL11, pUL12, and pUL13 in E. coli 1100 indicate that LipB is indispensable SOWX Protein Sequence for lipolytic activity in E. cob and that the terminus of ZipB lies between nucleotides 2248 (next to the Eco47111 His261 restriction site) and 2347 (the terminal nucleotide from P. aeruginosa M N H251 L D E TEI3235 the Pseudomonas host strain in pUL12). Jorgensen et al. WNH LDE P. cqacia DSM 3959 (13) showed that the lipase gene lipA from P. cepaciu DSM P. frogi IFO-12049 LDH LDT 3959 is expressed in heterologous hosts only when the Human Gastric lipase YNH LDF Rat Lingual lipane YNH LDF host strain contains the ZimA gene. Iizumi et al. (16) rePancreatic lipase CNH LRS Dog ported that in Pseudomonas sp. KWI-56, the act gene Pig Pancreatic lipase CNH LRS seven bases downstream of the end of the lipase gene, lip, Human Pancreatic lipase CNH LRS Rat Hepatic lipsse CA H E RS is needed for high lipase activity. Both ZimA and act code HUIIIEUI Hepatic lipase CS H E RS for 344 amino acid residues and are similar in size to the Guinea pig LPL CSH ERS 1ipB gene reported here (339 residues). The deduced amino Chicken LPL CS H E RS CS H E RS MOW LPL acid sequence of the 1ipB product was 27% identical to COW CSH ERS LPL those of limA and act products, although the amino acid HLPL CS H E RS A-209 sequences of the N-terminal region of the three genes NF L P. at?Nginosa D209 P S were less similar than this. E. coli 1100 cells transformed TE3235 with pUL12, in which the 1ipB product should have a NVL DLS P. cepoeio DSM 3959 NLL DPL P. fmgi IFO-12049 deletion of 21 amino acid residues at the C-terminal reTGL DAA Hepatic lipase HUIIX%l gion, had no lipolytic activity when grown on tributyrin TGL DAA Hepatic lipese Rabbit plates. This outcome of deletion experiments together TGL DPA Hepatic lipase Rat TGL DPA Hepatic lipase Mouse with the results of sequence alignment showed that the TGL DAA LPL Guinea pig C-terminal region of the ZipB product was functionally TG L D PA LPL Chicken important. For limA and act, functions that have been TG L D PA LPL M0Wie TG L D PA LPL COW postulated for this region are stimulation of elongation TGL DPA LPL Human or stabilization of the mRNA for the lipase or the gene TGL DPA Pancreatic lipaae MOU% product, facilitation of secretion, and processing or folding TG L D PA Pancreatic lipase Pig TG L D PA Pancreatic lipase HlUIlall of the lipase precursor (13, 16). TGL DPV Pancreatic lipase Dog Ihara et al. (41) have cloned 1ipL from Pseudomonas nov. sp. 109. It codes for the lactonizing lipase, which catalyzes the intramolecular transesterification of methyl that the carboxyl end of the tyrosine residue is not sus- 16-hydroxyhexadecanoate, giving cyclohexadecanolide. ceptible to trypsin, Tyr196 should be exposed to the sol- This lipase is similar to that from P. aeruginosa TE3285 vent and easily accessible to trypsin for cleavage; Tyr196 in their molecular masses estimated by SDS-PAGE (29 is on the loop and protrudes into the bulk solvent phase. and 28 kDa), p1 (5.3 and 5.95), optimum pH (7-10 and 7-9), and optimum temperature (45 and 4550°C). Amino The loop in bovine lipoprotein lipase is sensitive to tryptic digestion under mild conditions (40); it is cleaved at acid sequences deduced from the nucleotide sequence of Arg228, which is thought to be on a loop, to judge from 1ipL and 1ipA did not match at only six positions. The the results of sequence alignment with human pancreatic 2.2-kb DNA fragment containing 1ipL is required for the lipase. The residues next to the tryptic cleavage site, expression of activity in E. coli JM105, but the sequencing Trp198, are conserved among five lipases from Pseudo- of the whole of the fragment is not yet complete. In P. monas species. In human pancreatic lipase, Trp252 on aeruginosa TE3285, a 2.1-kb DNA fragment on pULl1 the loop is a functionally important residue that covers was needed for lipase activity. As described above, lipases the active serine residue. On the basis of these observa- from Pseudomonas nov. sp. 109 and P. aeruginosa TE3285, coded by 1ipL and lipA, respectively, have very similar tions, the region containing Trp198 in P. aeruginosa natures. It is not known if there is a related gene downTE3285 seemed to be a surface loop. The putative surface loop of the P. aeruginosa TE3285 stream of ZipL, but a similar gene might be expected in lipase is shorter by six residues than that of IFO-12049 this region. and shorter by 22 residues than that of the lipases from M-12-33, DSM 3959, and KWI-56. No large deletion or ACKNOWLEDGMENTS insertion was observed in any other region of the PseuWe thank Drs. R. Hayashi and B. Mikami, Research Institute for Food Science, Kyoto University, and Dr. A. Oka, Institute for Chemical domonas lipases. That only the lipase from P. aeruginosa TABLE
III
Comparisonof Amino Acid Sequencesof Several Lipases in the Vicinity of Asp209and His251 of P. aerugirwsa TE3285 Lipase
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Research, Kyoto University, for advice in amino acid analysis and sequencing of N-terminal amino acids and nucleotides; Dr. A. Matsuyama, Noda Institute for Scientific Research, for providing E. coli strain 1100; and Dr. T. Nakazawa, School of Medicine, Yamaguchi University, and Drs. M. Oka and B. Kawakami, Toyobo Co., Ltd., for discussions.
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