Biosci. Biotech. Biochem., 56 (12), 2074-2075, 1992
Note
Molecular Cloning and Structure of the Gene for Esterase from a Thermophilic Bacterium, Bacillus stearothermophilus IFO 12550 t Wataru
KUGIMIYA,
Yasuo
OTANI,
and Yukio
HASHIMOTO
Central Research Institute, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun, Ibaraki 300-24, Japan Received June 15, 1992
Carboxyl esterase (EC 3.1.1.1) catalyzes the hydrolysis of a large number of carboxylic esters and is widely distributed in various kinds of living organisms. 1 ) Recent interest in the use of esterases for the development of flavor in foodstuffs has generated an impetus to investigate sources and properties of microbial esterases. The esterase gene (est) from Bacillus stearothermophilus IFO 12550 was expressed in Bacillus brevis. 2) The esterase was characterized by a high thermostability, and a preference for triglycerides with short-chain fatty acids rather than long-chain ones. 2 ) This paper describes the molecular cloning and DNA sequence of the est gene.
2 3 4
B
The source of the gene was B. stearothermophilus IFO 12550 grown in L-broth at 55°C. The preparation of chromosomal DNA, and methods for cloning, nucleotide sequencing, and protein sequencing were done as previously described. 3.4) The chromosomal DNA from B. stearothermophilus was completely digested with HindIlI, inserted into the HindUI site of pUC9, and used to transform Escherichia coli JM83. From 10,000 transformants screened on tributyrin agar plates,3) a single halo-forming colony was obtained (see Fig. 1B). After preparation of the plasmid DNA (pBH7) from the halo-forming colony, a restriction map was constructed as shown in Fig. 2. Subcloning with selection based on the halo-forming activity located the est gene to a 1.7-kb Sa/l-Accl fragment of pBH7. To locate the est gene more closely, the 1.7-kb Sall-Accl fragment was extracted and digested with Bal 31 nuclease. DNA treated with Bal 31 nuclease for the appropriate time was repaired in an end-filling reaction using the Klenow fragment of E. coli DNA polymerase I. After ligation
AGCAGAAAGCACGCGGCGCTCGATGCGGTGTTTCAGCGCGTGACGGTTGTGTTGGCCGTT -121 TTGTTCTTTGTGTTGGCGATTCTTGTTGTTTATGTCCAACCATCATAAGCAAAAAACGGG -61 GGCGGTCCAAAGAGAAGCGGGCTGACCTTTTCTATTTCAG1tAG~AGGAAGGAGAACGA
... Fig. 1.
Esterase Production by Various Strains.
(A): SDS-PAGE of the heat-treated cell extract protein of (I) E. coli JM83 (pBC!), (2) E. coli JM83 (pBH7), (3) E. coli JM83 (pUC9), and (4) marker proteins. The following proteins were used as protein size markers; lysozyme from egg white (14,300), trypsin inhibitor from soybeans (21,500), and bovine serum albumin (67,000). (B): Expression of the est gene in E. coli on a tributyrin agar plate. 3 ) Upper, E. coli JM83 (pUC9); middle, E. coli JM83 (pBH7); lower E. coli JM83 (pBCI).
H
A
s
Fig. 2.
-1
pBC1
Restriction Map of pBH7.
The thin line represents the pUC9 moiety and the double line the B. stearothermophi/us DNA fragment. The coding region of the est gene is shown by the solid line. Cleavage sites of AccI, Sail, HindIII, and PstI are indicated by A, S, H, and P.
ATGATGAAAATTGTTCCGCCGAAGCCGTTTTTCTTTGAAGCCGGGGAGCGGGCGGTGCTG M M K I V P P K P F F F E AGE R A V L
60 20
CTGTTGCATGGGTTTACCGGCAATTCCGCTGATGTTCGGATGCTCGGGCGTTTTTTAGAA L L H G F T G N SAD V R M L G R F L E
120 40
TCCAAAGGCTATACGTGCCATGCGCCTATTTACAAAGGACACGGCGTGCCGCCTGAG GAG S K G Y T C HAP I Y K G H G V P PEE
180 60
CTCGTCCACACCGGGCCGGATGACTGGTGGCAAGATGTCATGAACGGCTACGAGTTTTTG L V H T GPO 0 W W Q 0 V M N G Y E F L
240 80
AAAAACAAGGGCTACGAAAAAATCGCCGTCGCCGGACTGTCGCTTGGAGGCGTATTTTCA K N K G Y E K I A V A G L [§] L G G V F S
300 100
TTGAAATTAGGTTACACTGTACCTATAGAGGGCATTGTGACGATGTGCGCGCCGATGTAC L K L G Y T V PIE G I V T M CAP M Y
360 120
ATCAAAAGCGAGGAAACGATGTACGAAGGCGTGCTCGAGTATGCGCGCGAGTATAAAAAG I K SEE T M Y E G V LEY ARE Y K K
420 140
CGGGAAGGAAAATCAGAGGAGCAGATCGAACAGGAGATGGAGAAGTTCAAGCAGACGCCG REG K SEE Q I E Q E M E K F K Q T P
480 160
ATGAAGACGTTAAAGGCGCTGCAGGAGCTGATCGCCGATGTGCGTGACCATCTTGATTTG M K T L K A L Q ELI A 0 V R 0 H L 0 L
540 180
ATTTATGCCCCGACGTTTGTTGTGCAGGCGCGCCATGATGAGATGATCAACCCGGACAGC I YAP T F V V Q A RHO E MIN P 0 S
600 200
GCGAACATCATTTATAACGAAATTGAATCGCCGGTCAAACAAATCAAGTGGTATGAGCAA A N I I Y N E I ESP V K Q I K W Y E Q
660 220
TCAGGCCATGTGATTACGCTTGATCAAGAAAAAGATCAGCTGCATGAAGATATTTATGCA S G H V I T L 0 Q E K 0 Q L H E 0 I Y A
720 240
TTTCTTGAATCGTTAGATTGGTAATGGAGAAAGGAGGGGATGGACAATGGATCATGTGTT P L E S LOW *
780 247
GGCCGAACGCATCTTACGTTTATGCGCGATGAGGCGTATAAGCCGATGACGGTCGAAGAG
840
Fig. 3. Gene.
Nucleotide and Deduced Amino Acid Sequences of the Est
A probable SD sequence and the deduced N-terminal sequences that were confirmed by the sequence data of the purified esterase are underlined. * indicates a stop codon. Ser94, which is believed to be the active site serine, is boxed.
t The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the following accession number: D12681.
NII-Electronic Library Service
Esterase Gene from Bacillus stearothermophilus with SmaI-cut pUC9, the DNA was used to transform E. coli JM83. One of the resulting transformants, E. coli JM83 harboring pBCl, formed a large halo as shown in Fig. lB. Each transformant was grown in 100 ml of L-broth containing ampicillin for 24 h. The cells were collected by centrifugation, washed once, and resuspended in 1.5 ml of 50 mM phosphate buffer (pH 8.0). A soluble extract was prepared by sonication followed by centrifugation of the suspension for 1 hat 20,000 x g. The cell extract was then incubated at 70°C for 10 min and centrifuged at 20,000 x 9 for 10 min. The resulting supernatants were electrophoresed by SDS~PAGE by the method of Laemmli. 5 ) A protein of about 29 kDa, absent in the cell extract of E. coli JM83 (pUC9), was present in large amounts in that of E. coli JM83 (pBCI) as shown in Fig. lA. This 29-kDa protein was also present in small amounts in the cell extract of E. coli JM83 (pBH7). The quantity of the 29-kDa protein in the cell extract of these transformants correlated with the size of the halo formed by these transformants. Further, the molecular weight of this protein was identical to that of the esterase derived from the est gene expressed in B. brevis. 2 ) Therefore, it was concluded that the est gene was cloned in E. coli and the 29-kDa protein was the active esterase. The 29-kDa protein could be purified to homogenity from the heat-treated cell extract of E. coli JM83 (pBCI) by one passage through a DEAE-Sephadex A50 column. The N-terminal amino acid sequence of the protein was found by Edman degradation to be Met-Lys-Ile-Val-ProPro-Lys-Pro-Phe-Phe-. The nucleotides were sequenced for 1.0 kb in the 1.7-kb Sall-Acel fragment, and are shown along with the deduced amino acid sequence in Fig. 3. There was only one large open reading frame in the 1020-bp nucleotide sequence, beginning with an ATG codon at nucleotide 1 and ending with a T AA codon at nucleotide 742. The N-terminal amino acid sequence of the 29-kDa protein was found at residues 2 to 11 as shown in Fig. 3. The molecular mass of the preprotein of 247 amino acids, formed by this open reading frame, was calculated to be 28,387. This data coincided well with a molecular mass of 29-kDa measured by the SDS-PAGE.
2075
Upstream from A TG, there was a putative ribosome-binding sequence, 5' -AGAAAGGAA-3' which is complementary to the 3' terminus of the 16S rRNA from B. stearothermophilus. 6 } The methionine was followed by a sequence of hydrophobic amino acids, and these may be the core of a signal sequence for secretion. The most probable cleavage site was predicted by the method of Perlman and Halvorson 7) to be between residues Gly24 and Phe25. In the case of pBCI, the 5' region of the est gene was deleted by treatment with Bal 31 nuclease as shown in Fig. 3. It may therefore be 'concluded that the enhanced expression of the est gene in pBCI was due to the transcription from a lacZ promoter of pUC9. This esterase is reported to be a serine enzyme since it was completely inhibited by organophosphorus compounds such as diisopropyl fiuorophosphate and phenylmethylsulfonyl fluoride. 2) The sequence G-X-S-X-G, which is the consensus active site sequence of serine esterase,8) was found at residues 92 to 96. Therefore Ser94 is postulated to be the active serine residue.
References 1)
K. Krisch, in "The Enzymes," Vol. 5, 3rd Ed., ed. by P. D. Boyer, Academic Press, New York, 1971, pp. 43-69. 2) Y. Amaki, E. E. Tutin, S. Ueda, K. Ohmiya, and T. Yamane, Biosci. Biotech. Biochem., 56, 238-241 (1992). 3) W. Kugimiya, Y. Otani, Y. Hashimoto, and Y. Takagi, Biochem. Biophys. Res. Commun., 141, 185-190 (1986). 4) W. Kugimiya, Y. Otani, M. Kohno, and Y. Hashimoto, Biosci. Biotech. Biochem., 56,716-719 (1992). 5) U. K. Laemmli, Nature, 227, 68()--'685 (1970). 6) M. Kozak, Microbiol. Rev., 47, 1-45 (1983). 7) D. Perlman and H. O. Halvorson,). Mol. Bioi., 167,391-409 (1983). 8) S. Brenner, Nature, 334, 528-530 (1988).
NII-Electronic Library Service
Note
Molecular Cloning and Structure of the Gene for Esterase from a Thermophilic Bacterium, Bacillus stearothermophilus IFO 12550 t Wataru
KUGIMIYA,
Yasuo
OTANI,
and Yukio
HASHIMOTO
Central Research Institute, Fuji Oil Co., Ltd., 4-3 Kinunodai, Yawara, Tsukuba-gun, Ibaraki 300-24, Japan Received June 15, 1992
Carboxyl esterase (EC 3.1.1.1) catalyzes the hydrolysis of a large number of carboxylic esters and is widely distributed in various kinds of living organisms. 1 ) Recent interest in the use of esterases for the development of flavor in foodstuffs has generated an impetus to investigate sources and properties of microbial esterases. The esterase gene (est) from Bacillus stearothermophilus IFO 12550 was expressed in Bacillus brevis. 2) The esterase was characterized by a high thermostability, and a preference for triglycerides with short-chain fatty acids rather than long-chain ones. 2 ) This paper describes the molecular cloning and DNA sequence of the est gene.
2 3 4
B
The source of the gene was B. stearothermophilus IFO 12550 grown in L-broth at 55°C. The preparation of chromosomal DNA, and methods for cloning, nucleotide sequencing, and protein sequencing were done as previously described. 3.4) The chromosomal DNA from B. stearothermophilus was completely digested with HindIlI, inserted into the HindUI site of pUC9, and used to transform Escherichia coli JM83. From 10,000 transformants screened on tributyrin agar plates,3) a single halo-forming colony was obtained (see Fig. 1B). After preparation of the plasmid DNA (pBH7) from the halo-forming colony, a restriction map was constructed as shown in Fig. 2. Subcloning with selection based on the halo-forming activity located the est gene to a 1.7-kb Sa/l-Accl fragment of pBH7. To locate the est gene more closely, the 1.7-kb Sall-Accl fragment was extracted and digested with Bal 31 nuclease. DNA treated with Bal 31 nuclease for the appropriate time was repaired in an end-filling reaction using the Klenow fragment of E. coli DNA polymerase I. After ligation
AGCAGAAAGCACGCGGCGCTCGATGCGGTGTTTCAGCGCGTGACGGTTGTGTTGGCCGTT -121 TTGTTCTTTGTGTTGGCGATTCTTGTTGTTTATGTCCAACCATCATAAGCAAAAAACGGG -61 GGCGGTCCAAAGAGAAGCGGGCTGACCTTTTCTATTTCAG1tAG~AGGAAGGAGAACGA
... Fig. 1.
Esterase Production by Various Strains.
(A): SDS-PAGE of the heat-treated cell extract protein of (I) E. coli JM83 (pBC!), (2) E. coli JM83 (pBH7), (3) E. coli JM83 (pUC9), and (4) marker proteins. The following proteins were used as protein size markers; lysozyme from egg white (14,300), trypsin inhibitor from soybeans (21,500), and bovine serum albumin (67,000). (B): Expression of the est gene in E. coli on a tributyrin agar plate. 3 ) Upper, E. coli JM83 (pUC9); middle, E. coli JM83 (pBH7); lower E. coli JM83 (pBCI).
H
A
s
Fig. 2.
-1
pBC1
Restriction Map of pBH7.
The thin line represents the pUC9 moiety and the double line the B. stearothermophi/us DNA fragment. The coding region of the est gene is shown by the solid line. Cleavage sites of AccI, Sail, HindIII, and PstI are indicated by A, S, H, and P.
ATGATGAAAATTGTTCCGCCGAAGCCGTTTTTCTTTGAAGCCGGGGAGCGGGCGGTGCTG M M K I V P P K P F F F E AGE R A V L
60 20
CTGTTGCATGGGTTTACCGGCAATTCCGCTGATGTTCGGATGCTCGGGCGTTTTTTAGAA L L H G F T G N SAD V R M L G R F L E
120 40
TCCAAAGGCTATACGTGCCATGCGCCTATTTACAAAGGACACGGCGTGCCGCCTGAG GAG S K G Y T C HAP I Y K G H G V P PEE
180 60
CTCGTCCACACCGGGCCGGATGACTGGTGGCAAGATGTCATGAACGGCTACGAGTTTTTG L V H T GPO 0 W W Q 0 V M N G Y E F L
240 80
AAAAACAAGGGCTACGAAAAAATCGCCGTCGCCGGACTGTCGCTTGGAGGCGTATTTTCA K N K G Y E K I A V A G L [§] L G G V F S
300 100
TTGAAATTAGGTTACACTGTACCTATAGAGGGCATTGTGACGATGTGCGCGCCGATGTAC L K L G Y T V PIE G I V T M CAP M Y
360 120
ATCAAAAGCGAGGAAACGATGTACGAAGGCGTGCTCGAGTATGCGCGCGAGTATAAAAAG I K SEE T M Y E G V LEY ARE Y K K
420 140
CGGGAAGGAAAATCAGAGGAGCAGATCGAACAGGAGATGGAGAAGTTCAAGCAGACGCCG REG K SEE Q I E Q E M E K F K Q T P
480 160
ATGAAGACGTTAAAGGCGCTGCAGGAGCTGATCGCCGATGTGCGTGACCATCTTGATTTG M K T L K A L Q ELI A 0 V R 0 H L 0 L
540 180
ATTTATGCCCCGACGTTTGTTGTGCAGGCGCGCCATGATGAGATGATCAACCCGGACAGC I YAP T F V V Q A RHO E MIN P 0 S
600 200
GCGAACATCATTTATAACGAAATTGAATCGCCGGTCAAACAAATCAAGTGGTATGAGCAA A N I I Y N E I ESP V K Q I K W Y E Q
660 220
TCAGGCCATGTGATTACGCTTGATCAAGAAAAAGATCAGCTGCATGAAGATATTTATGCA S G H V I T L 0 Q E K 0 Q L H E 0 I Y A
720 240
TTTCTTGAATCGTTAGATTGGTAATGGAGAAAGGAGGGGATGGACAATGGATCATGTGTT P L E S LOW *
780 247
GGCCGAACGCATCTTACGTTTATGCGCGATGAGGCGTATAAGCCGATGACGGTCGAAGAG
840
Fig. 3. Gene.
Nucleotide and Deduced Amino Acid Sequences of the Est
A probable SD sequence and the deduced N-terminal sequences that were confirmed by the sequence data of the purified esterase are underlined. * indicates a stop codon. Ser94, which is believed to be the active site serine, is boxed.
t The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases with the following accession number: D12681.
NII-Electronic Library Service
Esterase Gene from Bacillus stearothermophilus with SmaI-cut pUC9, the DNA was used to transform E. coli JM83. One of the resulting transformants, E. coli JM83 harboring pBCl, formed a large halo as shown in Fig. lB. Each transformant was grown in 100 ml of L-broth containing ampicillin for 24 h. The cells were collected by centrifugation, washed once, and resuspended in 1.5 ml of 50 mM phosphate buffer (pH 8.0). A soluble extract was prepared by sonication followed by centrifugation of the suspension for 1 hat 20,000 x g. The cell extract was then incubated at 70°C for 10 min and centrifuged at 20,000 x 9 for 10 min. The resulting supernatants were electrophoresed by SDS~PAGE by the method of Laemmli. 5 ) A protein of about 29 kDa, absent in the cell extract of E. coli JM83 (pUC9), was present in large amounts in that of E. coli JM83 (pBCI) as shown in Fig. lA. This 29-kDa protein was also present in small amounts in the cell extract of E. coli JM83 (pBH7). The quantity of the 29-kDa protein in the cell extract of these transformants correlated with the size of the halo formed by these transformants. Further, the molecular weight of this protein was identical to that of the esterase derived from the est gene expressed in B. brevis. 2 ) Therefore, it was concluded that the est gene was cloned in E. coli and the 29-kDa protein was the active esterase. The 29-kDa protein could be purified to homogenity from the heat-treated cell extract of E. coli JM83 (pBCI) by one passage through a DEAE-Sephadex A50 column. The N-terminal amino acid sequence of the protein was found by Edman degradation to be Met-Lys-Ile-Val-ProPro-Lys-Pro-Phe-Phe-. The nucleotides were sequenced for 1.0 kb in the 1.7-kb Sall-Acel fragment, and are shown along with the deduced amino acid sequence in Fig. 3. There was only one large open reading frame in the 1020-bp nucleotide sequence, beginning with an ATG codon at nucleotide 1 and ending with a T AA codon at nucleotide 742. The N-terminal amino acid sequence of the 29-kDa protein was found at residues 2 to 11 as shown in Fig. 3. The molecular mass of the preprotein of 247 amino acids, formed by this open reading frame, was calculated to be 28,387. This data coincided well with a molecular mass of 29-kDa measured by the SDS-PAGE.
2075
Upstream from A TG, there was a putative ribosome-binding sequence, 5' -AGAAAGGAA-3' which is complementary to the 3' terminus of the 16S rRNA from B. stearothermophilus. 6 } The methionine was followed by a sequence of hydrophobic amino acids, and these may be the core of a signal sequence for secretion. The most probable cleavage site was predicted by the method of Perlman and Halvorson 7) to be between residues Gly24 and Phe25. In the case of pBCI, the 5' region of the est gene was deleted by treatment with Bal 31 nuclease as shown in Fig. 3. It may therefore be 'concluded that the enhanced expression of the est gene in pBCI was due to the transcription from a lacZ promoter of pUC9. This esterase is reported to be a serine enzyme since it was completely inhibited by organophosphorus compounds such as diisopropyl fiuorophosphate and phenylmethylsulfonyl fluoride. 2) The sequence G-X-S-X-G, which is the consensus active site sequence of serine esterase,8) was found at residues 92 to 96. Therefore Ser94 is postulated to be the active serine residue.
References 1)
K. Krisch, in "The Enzymes," Vol. 5, 3rd Ed., ed. by P. D. Boyer, Academic Press, New York, 1971, pp. 43-69. 2) Y. Amaki, E. E. Tutin, S. Ueda, K. Ohmiya, and T. Yamane, Biosci. Biotech. Biochem., 56, 238-241 (1992). 3) W. Kugimiya, Y. Otani, Y. Hashimoto, and Y. Takagi, Biochem. Biophys. Res. Commun., 141, 185-190 (1986). 4) W. Kugimiya, Y. Otani, M. Kohno, and Y. Hashimoto, Biosci. Biotech. Biochem., 56,716-719 (1992). 5) U. K. Laemmli, Nature, 227, 68()--'685 (1970). 6) M. Kozak, Microbiol. Rev., 47, 1-45 (1983). 7) D. Perlman and H. O. Halvorson,). Mol. Bioi., 167,391-409 (1983). 8) S. Brenner, Nature, 334, 528-530 (1988).
NII-Electronic Library Service
