World
Journal
of Microbiology
& Biotechnology
12, 619-623
Cloning and nucleotide sequencing of a Iipase gene from Bacillus subtilis WRRL-B558 B.W. Thanomsub,
C. Boonchird
and V. Meevootisom’
A lipase gene from B. subtilis WRRL-B558 was cloned in Escherichia coli JMlO9 using pBluescript as a vector plasmid. Two methods were combined to screen for the lipase-producing clone. The first was done by overlaying the screening plates with j?-naphthylacetate and Fast Blue BB dye. Positive clones were then confirmed by a second method using 1% (v/v) tributyrin agar plates. Positive clones which formed clear zones on the tributyrin agar plates were selected and analysed by restriction mapping, Southern blot hybridization and deletion studies to locate the lipase gene on a 2.2 kb HindIII fragment insert. A subclone harbouring a plasmid with a 0.9 kb DNA fragment between the Hind111 and EcoRI sites that still exhibited lipase activity was used for sequencing. The nucleotide sequence showed a single open reading frame which contained 636 nucleotides (212 deduced amino acids). A conserved pentapeptide postulated to be the catalytic site was Ala-X-Ser-X-Gly instead of Gly-X-Ser-XGly. The deduced protein was found to have a molecular weight of 21 kDa which was similar to that obtained from the recombinant plasmid as determined by SDS-PAGE. Expression of the Bacillus lipase gene was found to be high in recombinant E. coli. Key words:
B. s&f&s, DNA
sequence, gene cloning, lipase.
Lipases (triacylglycerol acylhydrolase; EC 3.1.1.3) constitute a class of enzymes which are able to hydrolyse ester bonds of triacylglycerols at oil-water interfaces. Lipases, particularly those produced by microorganisms, have received much attention over the past decade, as they are relatively stable, catalyse a variety of reactions, and are substrate specific (Harwood 1989; Godtfredsen 1990). Those properties are of potential importance for diverse industrial applications, such as the production of cocoa butter-like fat from cheap palm oil (Harwood 1989). improvement of productivity in paper manufacturing (SkjoldJorgensen & Lange tool), and synthesis of a glucoside ester for use as a biosurfactant (Bjorkling et al. 1989; Ducret et al. 1995). Bacillus stlbfilis has long been used in industry for production of various proteins because it is non-pathogenic and because its physiology and genetics are extensively underB.W. Thanomsub and V. Meevootisom are with the Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand: fax (662) 246-3026. C. Boonchird is with the Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. * Corresponding author. @ 1996 Rapid
Science
Publishers
stood. In this paper we report the molecular lipase gene from B. sclbfilis WRRL-B558.
Materials
cloning
of a
and Methods
Reagents
Ampicillin, P-naphthylacetate, Fast Blue BB salt, tributyrin, p-nitrophenylpalmitate, X-gal and IPTG (Isopropyl-/I-o-thiogalactoside) were purchased from Sigma. Restriction endonucleases and other modifying enzymes were obtained from Boehringer Mannheim. Strains, Bacillus
F’lusmids subtilis
and Growth
Conditions
strain WRRL-B558 kindly provided by Dr L.K. Nakamura (Culture Collection at the National Center for Agricultural Utilization Research at 1815 N. University, Peoria, 1161604) was used as a source of the lipase gene. B. subtilis was grown in tryptic soy broth at 30°C and 200 rev/min. Escherichiu coli JM109 (Yanisch-Perron et al. 1985) was used as a host for plasmid amplification at 37°C. The cloning vector used was pBluescript (Stratagene). Gene Cloning B. subtilis WRRL-B558
chromosomal DNA was prepared as previ-
B. W. Thanomsub, C. Boonchird and V. Meevoofisom
H
Bx E
P
ES
E
PvH
denatured gel electrophoresis. Two SDS gels were run in parallel. One gel was stained with Coomassie Brilliant Blue, the other was renatured in 0.04 M Tris/HCl buffer pH 6.0, containing 0.5% (v/v) Triton X-100 overnight at 4°C and prewarmed for 30 min at room temperature in 0.1 M phosphate buffer pH 7.5 before being stained for lipase activity. Staining for lipase activity was done by soaking gels with shaking for 1 (native gels) or 6 (renatured SDS gels) h in 0.1 M phosphate buffer pH 7.5, containing 50 mg Fast Blue BB dye and 20 mg P-naphthylacetate (Higerd 1977).
Results
Flgure 1. represent respectively. of the lac EcoRI; l-l,
Physical map of plasmid pBL1. Thick and thin lines B. subtilis lipase DNA and vector pBluescript (PBS) The arrow indicates the direction of transcription promotor from PBS. Restriction enzymes: Bx, BstXI; E, Hindlll; P, Pstl; Pv. Pvull; S, Sall.
ously described (Miura 1967). The DNA was digested with Hi&II restriction enzyme and ligated with HindUI-digested and dephosphorylated pBluescript (PBS) vector. The ligation mixture was introduced into competent E. coli JM109 (Mandel & Higa 1970). Transformants were screened by overlaying with /I-naphthylacetate and Fast Blue BB dye (Higerd 1977). Positive clones were then confirmed by replicating on I% (v/v) tributyrin agar plates (Kugimiya et al. 1986). The colonies producing clear zones were selected and their recombinant plasmids were analysed by restriction mapping. Deletion Studies and Nucleotide Sequencing Subcloning experiments were performed to identify the smallest region of the inserted DNA necessary for production of the lipase. The DNA subclones were constructed according to restriction sites and tested for their abilities to hydrolyse tributyrin as described above. The DNA fragment encoding the lipase gene was sequenced by the dideoxy chain-termination method using a T7 sequencing kit (Pharmacia). The sequence data were analysed for the location of the open reading frame and its amino acid sequence was deduced with the DNASIS Software program (Hitachi Software Engineering Co. Ltd.). Enzyme Assay Lipase activity was determined in duplicate for each sample by the spectrophotometric method of Kordel et al. (1991). Enzyme activity was expressed as specific activity (nmol p-nitrophenol released/min/mg protein). Cells from a 5 ml overnight culture were washed and resuspended in 50 mM Tris/HCl pH 7.5. The cell suspension was treated with ultrasonic disintegration and its lipase activity was measured. The protein content of each extract was determined by the Lowry method.
Gel Electrophoresis Samples of sonicated cells were loaded onto 7.5% polyacrylamide gels for native gel electrophoresis and onto 15% SDS polyacrylamide gels containing 5 M urea and 0.5% (v/v) Triton X-100 for
620
World Journal of Microbiology 6 Biotechnology, Vol 12, 1996
and Discussion
Cloning the Lipase Gene from B. subtilis WRRLB558 Of the 20,000 colonies screened, 2 transformants were found to produce halo zones on the overlaid P-naphthylacetate agar and also to form clear zones on I% tributyrin agar plates. The recombinant plasmids, each containing a 2.2 kb fragment of inserted DNA, were recovered from the transformants and were designated pBL1 and pBL2. Restriction analysis showed that pBL1 and pBL2 were similar to each other but different in orientation. Therefore, only the restriction map of pBL1 is shown in Figure 1. Deletion Sfudies and Sequencing As shown in Figure 2, the lipase gene was probably located on the HindIII-EcoRI fragment whose size was about 0.9 kb. This DNA fragment was subjected to DNA sequencing; the strategy used is shown in Figure 3. The nucleotide sequence of the HindIII-EcoRI fragment contained a single open reading frame of 636 nucleotides. The deduced primary structure of the lipase showed that it was composed of 212 amino acid residues. The molecular mass calculated from the deduced amino acid sequence was 22,693 Da. This lipase gene sequence lacked the conserved pentapeptide Gly-X-Ser-X-Gly which is thought to be characteristic of the catalytic site of lipase enzymes. Instead, it had the sequence Ala-His-Ser-Met-Gly, which was similar to the conserved peptide except that the first Gly was substituted by Ala. It is possible that this pentapeptide confers the catalytic function of the lipase enzyme. This sequence was identical to that of lipases from another B. subtilis and B. pwnilus (Dartois et al. 1992) strains where it has also been hypothesized to constitute the catalytic site. Southern blot hybridization of HindIII-digested B. stcbtilis DNA with a 0.7 kb probe containing a HindIII-BsfXI fragment of pBL1 (underlined in Figure 4) showed a single positive band of 2.2 kb (data not shown). The result suggested that the cloned lipase gene was present as a single copy in B. w&f&s WRRL-B558. Expression of fhe Lipase Gene from B. subtilis in E. coli The lipase activity of E. coli harbouring pBL1 containing the lipase gene from B. strbfilis WRRL-B558 was determined by comparison with that harbouring only the pBluescript
Cloning of B. subtilis lipuse gene
Lipase activity (Clear zone on Tributyrin agar plate) H
P
BxE
ES
E PvH
H
Bx
H
100bp
Figure 2. Deletion study of the DNA encoding B. subtilis lipase from pBL1. The subclones containing DNA fragments, as indicated the bars, were checked for the lipolytic activity phenotype by clear zone production on 1% (v/v) tributyrin agar plates. Abbreviations of restriction enzymes: Bx, BstXI; E, EcoRI; H, Hindlll; P. Pstl; Pv, Pvull; S, Sall. Lipase activity: +, with a clear zone; -, without clear zone on tributyrin agar plates. H
P
BXE
E
Table 1. Lipase activlty 6. subfilis WRRL-B558 OrganismlPlasmid
Figure 3. Sequencing strategy of the 0.9 kb Hindlll-EcoRI DNA fragment on PBS. Different deletions of the 0.9 kb insert were generated using a restriction site in the polycloning site and another within the insert. The arrows indicate the nucleotide sequence obtained for each subclone, using a universal primer (leftward arrows) and reverse primer (rightward arrows). Thick arrows represent sequences derived using oligonucleotide primers synthesized from known sequences. Abbreviations of restriction enzymes: Bx, BstXI; E. EcoRI; H, Hindlll.
vector plasmid as a control. The result (Table 1) showed a high level of lipase production as expressed in unit/ min/mg protein. Enzyme activity was much higher with E. co/i harbouring the vector containing the gene insert than with E. co/i harbouring the vector only (p < 0.001 with Z-test) or with B. subtilis WRRL-B558 (p < 0.001 with Z-test). Gel Electrophoresis Activity staining of renatured
SDS gels using the chromo-
of 15 co/i JMlO9
carrying
plasmids
in a
and
Lipase Activity’ (nmol/mln/mg protein)
JMlOS/pBS JMlOS/pBLl JM109/pBL2 6. subtilis WRRL-B558
0.966 61.24 54.42 3.042
+ f + t
0.27=t 7.06b 9Mb 1.04c
’ The values shown are the means of 6 samples (3 experiments with 2 replicates each). T Mean values with different superscripts were significantly different (p 0.05) by the Z-test.
genie esterase substrate P-naphthylacetate showed that extracts from recombinant clones of E. coli had a single band for lipase activity (approximate 20 kDa) in addition to the two esterase/lipase bands (approximately 40 and 45 kDa) that were present with extracts from non-transformed E. co/i (Figure 5). Unexpectedly, no lipase band was detected at 20 kDa in extracts from B. subtilis, the donor strain although it did give a strong esterase band at 66 kDa as expected (Abbot & Fukuda 1975). This could have been due to the fact that the lipase activity was very low, as measured in tube assays with lipase substrate p-nitrophenylpalmitate (PNPP) (Table I).
World Journal
of
Microbiology 6 Biotechnology, Vol 12. 19%
621
B. W.
i’hnomsub, C. Boonchird 1
and V. Meevootisom
Hindm Ah %I!
TAT
TTC
MT
QAQ
TAT
GCT
TTT
TAG
40
TTT
TTT
TTG
96
TCT
TTA
AAA ATA
GTA
ATA
1 144
3
l *
AAA
QAA
GGT W-A
TM
GCC
TTC
47
GAC CM
TAA
TQA
CCT
CTG MT
CTT
AAA
ATT
95
AGC CM
AAT
TAC
CCT
TTC
CTT
AAT
TAA
TTT
GGT
AAC
143
TTG
TTA
CAA
AAA
AAG
AAA
FAT
ATT
t-4 K ATG AAA
191
I ATT
A I, GCQ CTT
V GTA
T ACA
I ATT
L TTG
H ATG
L CTG
S TCT
l6 239
A GCA
A
LL.
t
TAA
TTG
GAG AAT
K R AAA AGA
AGO ATC
F
V
192
TTT
GTA
19
T
240
V OTT
35 286
AAT
51 336
A G I GCG GGA ATT
K AA0
67 304 83 432 99
AGA
R
I
TQA
2
TTT
PstI L Q GCQ CTQ CAG
P CC0
TCA
K A+lj
GCC
A GCT
E GM
II CAC
287
ATG
OTT
CAC
GOT
ATG
GGA GGG GCA
TCA
TTC
PAT
TTT
50 335
S AGC
Y TAT
L CTC
V GTA
3 TCT
Q CAG
G GGC
S P TCG CCG
D K GAC MG
66 363
LYAVDFWDKTGTNYNN CTQ TAT GCA OTT
GAT
TTT
TGQ GAC AAQ
ACA
GGG ACA
AAT
TAT
AAC
431
G p V GGC CCQ QTA
S TCA
R CGA
V QTQ
Q CAA
D
0 GM
T
G
TTT
ACG
GOT
479
I ATT
V OTC
A
H
H ATG
G
G
GG#
GGC
A GCG
N MC
T
7
ACA
114 527
AAA AAT
CTG
GAC GGC GGA AAT
AAA
GTT
GCA
MC
GTC
130 575
L CTG
NPVVMVHGtdGGASFNF CCA OTC OTT
A
ACA
S TCA
K
L TTA
F
A
S
W TGF
34
82
K
V
D
AAA
GTQ
GAT
F
K V AAG GTT
S
L TTA
GAT
AAT
98
460
QCQ AAA
115
526
LYYIKNLDGGNKVANV CTT TAC TAC ATA
131 576
VTVQGANRLTTGKALP GTG ACG GTT GGC GGC GCG AAC COT
TTG
AC0
ACA
GGC AA0
GCG CTT
CCG
623
147 624
GTDPNQKILYTSIYSS GQA ACA GAT CCA MT
ATT
TTA
TAC
ACA
TCC
ATT
TAC
AGC
AGT
162 671
163 672
ADMIVMNYLSRLDGAR QCC GAT ATQ ATT
TAC
TTA
TCA
AGA
TTA
GAC
GOT
GCT AGA
178 719
G
N
G L GGC CTT
L CTG
Y TAC
9 9 AGC AGC
194 767
179
N
V
Q cAF,
720 195
QVNSLIKEGLNGGGQN CAR GTC ARC AGC
766 211
T
N
OTC ATG
mtx1 I H ATC CAT
ARC GTT
CAR MO
MT
0 G GGC GGT
146
GGA CAC
I ATC
210 CTG ATT
AAA GAA GGG CTG MC
GGC GGG GGC CAG MT
815 213
., BcoRI
Flgure4.
Southern
622
AAA
ACA AAA CCT
TGA
AGA ATT
CTA TTC TTC
Journal
of Microbiology
& Biotechnology
12, 619-623
Cloning and nucleotide sequencing of a Iipase gene from Bacillus subtilis WRRL-B558 B.W. Thanomsub,
C. Boonchird
and V. Meevootisom’
A lipase gene from B. subtilis WRRL-B558 was cloned in Escherichia coli JMlO9 using pBluescript as a vector plasmid. Two methods were combined to screen for the lipase-producing clone. The first was done by overlaying the screening plates with j?-naphthylacetate and Fast Blue BB dye. Positive clones were then confirmed by a second method using 1% (v/v) tributyrin agar plates. Positive clones which formed clear zones on the tributyrin agar plates were selected and analysed by restriction mapping, Southern blot hybridization and deletion studies to locate the lipase gene on a 2.2 kb HindIII fragment insert. A subclone harbouring a plasmid with a 0.9 kb DNA fragment between the Hind111 and EcoRI sites that still exhibited lipase activity was used for sequencing. The nucleotide sequence showed a single open reading frame which contained 636 nucleotides (212 deduced amino acids). A conserved pentapeptide postulated to be the catalytic site was Ala-X-Ser-X-Gly instead of Gly-X-Ser-XGly. The deduced protein was found to have a molecular weight of 21 kDa which was similar to that obtained from the recombinant plasmid as determined by SDS-PAGE. Expression of the Bacillus lipase gene was found to be high in recombinant E. coli. Key words:
B. s&f&s, DNA
sequence, gene cloning, lipase.
Lipases (triacylglycerol acylhydrolase; EC 3.1.1.3) constitute a class of enzymes which are able to hydrolyse ester bonds of triacylglycerols at oil-water interfaces. Lipases, particularly those produced by microorganisms, have received much attention over the past decade, as they are relatively stable, catalyse a variety of reactions, and are substrate specific (Harwood 1989; Godtfredsen 1990). Those properties are of potential importance for diverse industrial applications, such as the production of cocoa butter-like fat from cheap palm oil (Harwood 1989). improvement of productivity in paper manufacturing (SkjoldJorgensen & Lange tool), and synthesis of a glucoside ester for use as a biosurfactant (Bjorkling et al. 1989; Ducret et al. 1995). Bacillus stlbfilis has long been used in industry for production of various proteins because it is non-pathogenic and because its physiology and genetics are extensively underB.W. Thanomsub and V. Meevootisom are with the Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand: fax (662) 246-3026. C. Boonchird is with the Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. * Corresponding author. @ 1996 Rapid
Science
Publishers
stood. In this paper we report the molecular lipase gene from B. sclbfilis WRRL-B558.
Materials
cloning
of a
and Methods
Reagents
Ampicillin, P-naphthylacetate, Fast Blue BB salt, tributyrin, p-nitrophenylpalmitate, X-gal and IPTG (Isopropyl-/I-o-thiogalactoside) were purchased from Sigma. Restriction endonucleases and other modifying enzymes were obtained from Boehringer Mannheim. Strains, Bacillus
F’lusmids subtilis
and Growth
Conditions
strain WRRL-B558 kindly provided by Dr L.K. Nakamura (Culture Collection at the National Center for Agricultural Utilization Research at 1815 N. University, Peoria, 1161604) was used as a source of the lipase gene. B. subtilis was grown in tryptic soy broth at 30°C and 200 rev/min. Escherichiu coli JM109 (Yanisch-Perron et al. 1985) was used as a host for plasmid amplification at 37°C. The cloning vector used was pBluescript (Stratagene). Gene Cloning B. subtilis WRRL-B558
chromosomal DNA was prepared as previ-
B. W. Thanomsub, C. Boonchird and V. Meevoofisom
H
Bx E
P
ES
E
PvH
denatured gel electrophoresis. Two SDS gels were run in parallel. One gel was stained with Coomassie Brilliant Blue, the other was renatured in 0.04 M Tris/HCl buffer pH 6.0, containing 0.5% (v/v) Triton X-100 overnight at 4°C and prewarmed for 30 min at room temperature in 0.1 M phosphate buffer pH 7.5 before being stained for lipase activity. Staining for lipase activity was done by soaking gels with shaking for 1 (native gels) or 6 (renatured SDS gels) h in 0.1 M phosphate buffer pH 7.5, containing 50 mg Fast Blue BB dye and 20 mg P-naphthylacetate (Higerd 1977).
Results
Flgure 1. represent respectively. of the lac EcoRI; l-l,
Physical map of plasmid pBL1. Thick and thin lines B. subtilis lipase DNA and vector pBluescript (PBS) The arrow indicates the direction of transcription promotor from PBS. Restriction enzymes: Bx, BstXI; E, Hindlll; P, Pstl; Pv. Pvull; S, Sall.
ously described (Miura 1967). The DNA was digested with Hi&II restriction enzyme and ligated with HindUI-digested and dephosphorylated pBluescript (PBS) vector. The ligation mixture was introduced into competent E. coli JM109 (Mandel & Higa 1970). Transformants were screened by overlaying with /I-naphthylacetate and Fast Blue BB dye (Higerd 1977). Positive clones were then confirmed by replicating on I% (v/v) tributyrin agar plates (Kugimiya et al. 1986). The colonies producing clear zones were selected and their recombinant plasmids were analysed by restriction mapping. Deletion Studies and Nucleotide Sequencing Subcloning experiments were performed to identify the smallest region of the inserted DNA necessary for production of the lipase. The DNA subclones were constructed according to restriction sites and tested for their abilities to hydrolyse tributyrin as described above. The DNA fragment encoding the lipase gene was sequenced by the dideoxy chain-termination method using a T7 sequencing kit (Pharmacia). The sequence data were analysed for the location of the open reading frame and its amino acid sequence was deduced with the DNASIS Software program (Hitachi Software Engineering Co. Ltd.). Enzyme Assay Lipase activity was determined in duplicate for each sample by the spectrophotometric method of Kordel et al. (1991). Enzyme activity was expressed as specific activity (nmol p-nitrophenol released/min/mg protein). Cells from a 5 ml overnight culture were washed and resuspended in 50 mM Tris/HCl pH 7.5. The cell suspension was treated with ultrasonic disintegration and its lipase activity was measured. The protein content of each extract was determined by the Lowry method.
Gel Electrophoresis Samples of sonicated cells were loaded onto 7.5% polyacrylamide gels for native gel electrophoresis and onto 15% SDS polyacrylamide gels containing 5 M urea and 0.5% (v/v) Triton X-100 for
620
World Journal of Microbiology 6 Biotechnology, Vol 12, 1996
and Discussion
Cloning the Lipase Gene from B. subtilis WRRLB558 Of the 20,000 colonies screened, 2 transformants were found to produce halo zones on the overlaid P-naphthylacetate agar and also to form clear zones on I% tributyrin agar plates. The recombinant plasmids, each containing a 2.2 kb fragment of inserted DNA, were recovered from the transformants and were designated pBL1 and pBL2. Restriction analysis showed that pBL1 and pBL2 were similar to each other but different in orientation. Therefore, only the restriction map of pBL1 is shown in Figure 1. Deletion Sfudies and Sequencing As shown in Figure 2, the lipase gene was probably located on the HindIII-EcoRI fragment whose size was about 0.9 kb. This DNA fragment was subjected to DNA sequencing; the strategy used is shown in Figure 3. The nucleotide sequence of the HindIII-EcoRI fragment contained a single open reading frame of 636 nucleotides. The deduced primary structure of the lipase showed that it was composed of 212 amino acid residues. The molecular mass calculated from the deduced amino acid sequence was 22,693 Da. This lipase gene sequence lacked the conserved pentapeptide Gly-X-Ser-X-Gly which is thought to be characteristic of the catalytic site of lipase enzymes. Instead, it had the sequence Ala-His-Ser-Met-Gly, which was similar to the conserved peptide except that the first Gly was substituted by Ala. It is possible that this pentapeptide confers the catalytic function of the lipase enzyme. This sequence was identical to that of lipases from another B. subtilis and B. pwnilus (Dartois et al. 1992) strains where it has also been hypothesized to constitute the catalytic site. Southern blot hybridization of HindIII-digested B. stcbtilis DNA with a 0.7 kb probe containing a HindIII-BsfXI fragment of pBL1 (underlined in Figure 4) showed a single positive band of 2.2 kb (data not shown). The result suggested that the cloned lipase gene was present as a single copy in B. w&f&s WRRL-B558. Expression of fhe Lipase Gene from B. subtilis in E. coli The lipase activity of E. coli harbouring pBL1 containing the lipase gene from B. strbfilis WRRL-B558 was determined by comparison with that harbouring only the pBluescript
Cloning of B. subtilis lipuse gene
Lipase activity (Clear zone on Tributyrin agar plate) H
P
BxE
ES
E PvH
H
Bx
H
100bp
Figure 2. Deletion study of the DNA encoding B. subtilis lipase from pBL1. The subclones containing DNA fragments, as indicated the bars, were checked for the lipolytic activity phenotype by clear zone production on 1% (v/v) tributyrin agar plates. Abbreviations of restriction enzymes: Bx, BstXI; E, EcoRI; H, Hindlll; P. Pstl; Pv, Pvull; S, Sall. Lipase activity: +, with a clear zone; -, without clear zone on tributyrin agar plates. H
P
BXE
E
Table 1. Lipase activlty 6. subfilis WRRL-B558 OrganismlPlasmid
Figure 3. Sequencing strategy of the 0.9 kb Hindlll-EcoRI DNA fragment on PBS. Different deletions of the 0.9 kb insert were generated using a restriction site in the polycloning site and another within the insert. The arrows indicate the nucleotide sequence obtained for each subclone, using a universal primer (leftward arrows) and reverse primer (rightward arrows). Thick arrows represent sequences derived using oligonucleotide primers synthesized from known sequences. Abbreviations of restriction enzymes: Bx, BstXI; E. EcoRI; H, Hindlll.
vector plasmid as a control. The result (Table 1) showed a high level of lipase production as expressed in unit/ min/mg protein. Enzyme activity was much higher with E. co/i harbouring the vector containing the gene insert than with E. co/i harbouring the vector only (p < 0.001 with Z-test) or with B. subtilis WRRL-B558 (p < 0.001 with Z-test). Gel Electrophoresis Activity staining of renatured
SDS gels using the chromo-
of 15 co/i JMlO9
carrying
plasmids
in a
and
Lipase Activity’ (nmol/mln/mg protein)
JMlOS/pBS JMlOS/pBLl JM109/pBL2 6. subtilis WRRL-B558
0.966 61.24 54.42 3.042
+ f + t
0.27=t 7.06b 9Mb 1.04c
’ The values shown are the means of 6 samples (3 experiments with 2 replicates each). T Mean values with different superscripts were significantly different (p 0.05) by the Z-test.
genie esterase substrate P-naphthylacetate showed that extracts from recombinant clones of E. coli had a single band for lipase activity (approximate 20 kDa) in addition to the two esterase/lipase bands (approximately 40 and 45 kDa) that were present with extracts from non-transformed E. co/i (Figure 5). Unexpectedly, no lipase band was detected at 20 kDa in extracts from B. subtilis, the donor strain although it did give a strong esterase band at 66 kDa as expected (Abbot & Fukuda 1975). This could have been due to the fact that the lipase activity was very low, as measured in tube assays with lipase substrate p-nitrophenylpalmitate (PNPP) (Table I).
World Journal
of
Microbiology 6 Biotechnology, Vol 12. 19%
621
B. W.
i’hnomsub, C. Boonchird 1
and V. Meevootisom
Hindm Ah %I!
TAT
TTC
MT
QAQ
TAT
GCT
TTT
TAG
40
TTT
TTT
TTG
96
TCT
TTA
AAA ATA
GTA
ATA
1 144
3
l *
AAA
QAA
GGT W-A
TM
GCC
TTC
47
GAC CM
TAA
TQA
CCT
CTG MT
CTT
AAA
ATT
95
AGC CM
AAT
TAC
CCT
TTC
CTT
AAT
TAA
TTT
GGT
AAC
143
TTG
TTA
CAA
AAA
AAG
AAA
FAT
ATT
t-4 K ATG AAA
191
I ATT
A I, GCQ CTT
V GTA
T ACA
I ATT
L TTG
H ATG
L CTG
S TCT
l6 239
A GCA
A
LL.
t
TAA
TTG
GAG AAT
K R AAA AGA
AGO ATC
F
V
192
TTT
GTA
19
T
240
V OTT
35 286
AAT
51 336
A G I GCG GGA ATT
K AA0
67 304 83 432 99
AGA
R
I
TQA
2
TTT
PstI L Q GCQ CTQ CAG
P CC0
TCA
K A+lj
GCC
A GCT
E GM
II CAC
287
ATG
OTT
CAC
GOT
ATG
GGA GGG GCA
TCA
TTC
PAT
TTT
50 335
S AGC
Y TAT
L CTC
V GTA
3 TCT
Q CAG
G GGC
S P TCG CCG
D K GAC MG
66 363
LYAVDFWDKTGTNYNN CTQ TAT GCA OTT
GAT
TTT
TGQ GAC AAQ
ACA
GGG ACA
AAT
TAT
AAC
431
G p V GGC CCQ QTA
S TCA
R CGA
V QTQ
Q CAA
D
0 GM
T
G
TTT
ACG
GOT
479
I ATT
V OTC
A
H
H ATG
G
G
GG#
GGC
A GCG
N MC
T
7
ACA
114 527
AAA AAT
CTG
GAC GGC GGA AAT
AAA
GTT
GCA
MC
GTC
130 575
L CTG
NPVVMVHGtdGGASFNF CCA OTC OTT
A
ACA
S TCA
K
L TTA
F
A
S
W TGF
34
82
K
V
D
AAA
GTQ
GAT
F
K V AAG GTT
S
L TTA
GAT
AAT
98
460
QCQ AAA
115
526
LYYIKNLDGGNKVANV CTT TAC TAC ATA
131 576
VTVQGANRLTTGKALP GTG ACG GTT GGC GGC GCG AAC COT
TTG
AC0
ACA
GGC AA0
GCG CTT
CCG
623
147 624
GTDPNQKILYTSIYSS GQA ACA GAT CCA MT
ATT
TTA
TAC
ACA
TCC
ATT
TAC
AGC
AGT
162 671
163 672
ADMIVMNYLSRLDGAR QCC GAT ATQ ATT
TAC
TTA
TCA
AGA
TTA
GAC
GOT
GCT AGA
178 719
G
N
G L GGC CTT
L CTG
Y TAC
9 9 AGC AGC
194 767
179
N
V
Q cAF,
720 195
QVNSLIKEGLNGGGQN CAR GTC ARC AGC
766 211
T
N
OTC ATG
mtx1 I H ATC CAT
ARC GTT
CAR MO
MT
0 G GGC GGT
146
GGA CAC
I ATC
210 CTG ATT
AAA GAA GGG CTG MC
GGC GGG GGC CAG MT
815 213
., BcoRI
Flgure4.
Southern
622
AAA
ACA AAA CCT
TGA
AGA ATT
CTA TTC TTC
