Download Staphylococcus haemolyticus lipase

FEMS Microbiology Letters 179 (1999) 385^392
Staphylococcus haemolyticus lipase: biochemical properties,
substrate speci¢city and gene cloning
Byung-Chul Oh a , Hyung-Kwoun Kim a , Jung-Kee Lee a , Sun-Chul Kang b ,
Tae-Kwang Oh a; *
a
Microbial Enzyme Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejon 305-600,
South Korea
b
Department of Biotechnology, Taegu University, Taegu 120-749, South Korea
Received 1 June 1999 ; received in revised form 23 August 1999 ; accepted 25 August 1999
Abstract
Lipase of Staphylococcus haemolyticus L62 was purified from culture supernatant and its molecular mass was estimated to be
45 kDa by SDS-PAGE. Its optimum temperature and pH for the hydrolysis of olive oil was 28³C and pH 8.5, respectively. The
enzyme was stable up to 50³C in the presence of Ca2‡ and over the pH range 5^11. It had high hydrolytic activity against
tributyrin, tripropionin, and trimyristin among various triglycerides. The gene encoding the lipase was cloned in Escherichia
coli. Sequence analysis showed an open reading frame of 2136 bp, which encodes a preproenzyme of 711 amino acids. The
preproenzyme is composed of a signal peptide (60 aa), a pro-peptide (259 aa), and a mature enzyme (392 aa). The mature
enzyme has 49^67% amino acid sequence homology with other staphylococcal lipases. ß 1999 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Lipase; Staphylococcus haemolyticus ; Triglyceride ; Sequence homology
1. Introduction
Lipases are a class of enzymes that catalyze the
hydrolysis of long chain triacylglycerols. Many lipases have industrially important properties such as
chain length selectivity, regiospeci¢city, and chiral
selectivity. Therefore, the enzymes are often used
for the production of free fatty acids, interesteri¢cation of fats and oils, and synthesis of useful esters
and peptides.
* Corresponding author. Tel.: +82 (42) 860 4370;
Fax: +82 (42) 860 4595; E-mail: [email protected]
Many staphylococci are able to produce extracellular lipases and some of them have been puri¢ed
and their biochemical properties studied in detail
[1,2]. In contrast to most other lipases, some staphylococcal lipases are distinguished by their extremely
broad substrate speci¢city. Staphylococcus hyicus lipase (SHL) hydrolyzes neutral lipids almost irrespective of their chain length. In addition, it has a high
phospholipase activity, which distinguishes the enzyme not only from other staphylococcal lipases
but also from all bacterial lipases.
Staphylococcal lipase genes have been identi¢ed
from S. aureus [3,4], S. epidermidis [5,6], and S. hyicus [7]. These enzymes are produced as preproen-
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 3 9 - 5
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zyme forms, which have molecular masses of approximately 80 kDa. After secretion into the growth
medium, proteolytic processing results in mature
forms with molecular masses of 40^46 kDa [8].
Many comparative studies of S. aureus lipase
(SAL) and SHL have been done and in spite of
high homology (50^51%) in amino acid sequences,
they have found remarkable di¡erences in pH optimum, Ca2‡ a¤nity, and substrate speci¢city [4,9]. In
vivo chimeragenesis using SAL and SHL revealed
that phospholipase activity in SHL was determined
by three regions in its C-terminal domain and that
chain length selectivity was de¢ned by elements within the region of residues 180^250 in SHL [10].
We isolated a S. haemolyticus strain producing an
extracellular lipase from sewage treatment plants in
Korea. In this paper the lipase enzyme was puri¢ed
and its biochemical properties were studied. In addition, its corresponding gene was cloned and sequenced. We also compared its substrate speci¢city
and amino acid sequence with other staphylococcal
lipases.
2. Materials and methods
2.1. Puri¢cation of the lipase L62
S. haemolyticus L62 was cultivated in LB medium
(1% tryptone, 0.5% yeast extract, and 0.5% NaCl) at
37³C for 20 h. After ammonium sulfate was added to
the culture supernatant to 80% saturation, the precipitate was collected by centrifugation at 12 000Ug
for 30 min, dissolved in Tris-HCl bu¡er (20 mM, pH
8.0), and then dialyzed against the same bu¡er. The
dialysate was applied to a DEAE-Sepharose CL-6B
column (2.0U20 cm) pre-equilibrated with the same
bu¡er. Although most proteins in the dialysate
bound to the resin, lipase enzyme passed through
the column. The active fractions were collected and
re-loaded to a CM-Sepharose CL-6B column
(1.0U20 cm) pre-equilibrated with the same Tris
bu¡er. The bound proteins were eluted with a linear
gradient of NaCl (0^0.5 M) in the same bu¡er. The
active fractions were pooled, desalted, and then applied to a Resource S column (Pharmacia Biotech,
Uppsala, Sweden) pre-equilibrated with 20 mM Tris
bu¡er (pH 7.8). The lipase enzyme was eluted with a
0.3^0.4 M linear gradient of NaCl in the same bu¡er,
concentrated, and stored at 320³C.
2.2. Lipase assay
Lipase activity was measured by titrating free fatty
acids released by hydrolysis of olive oil using the pHstat method [11]. Olive oil emulsion was prepared by
emulsifying 5 ml of olive oil in 495 ml of 20 mM
NaCl, 1 mM CaCl2 , and 0.5% (w/v) gum arabic solution for 2 min at maximum speed in a Waring
blender. After the pH of the substrate emulsion (20
ml) was adjusted to 8.5 by the addition of 10 mM
NaOH solution, an appropriate amount (10^50 Wl)
of the enzyme solution was added. The release rate
of the fatty acid was measured with a pH titrator
(718 Stat Titrino, Metrohm, Switzerland) for 5 min
at 28³C. One lipase unit is de¢ned as the amount of
enzyme liberating 1 Wmol of fatty acid per minute.
Lipase activity for p-nitrophenyl esters was measured spectrophotometrically [12,13]. One milliliter of
p-nitrophenyl esters (C2 ^C12 , 10 mM in acetonitrile)
was mixed with ethanol (4 ml) and 95 ml of 50 mM
Tris-HCl (pH 8.5). An appropriate amount of the
lipase was added to 1 ml of this freshly prepared
substrate solution and the OD405 was measured after
3 min incubation at 28³C.
For long chain p-nitrophenyl esters (C12 ^C18 ),
20 Wl of lipase solution was added to 880 Wl of reaction bu¡er containing 50 mM Tris-HCl (pH 8.5),
0.1% gum arabic, and 0.2% deoxycholate. After
3 min incubation at 28³C, the reaction was initiated
by adding 100 Wl of 8 mM substrate in isopropanol.
The reaction was stopped by the addition of 0.5 ml
of 3 M HCl. After centrifugation, 333 Wl of supernatant was mixed with 1 ml of 2 M NaOH and the
OD420 was measured. One unit of lipase activity was
de¢ned as the amount of enzyme liberating 1 Wmol
of p-nitrophenol per minute.
Lipolytic activity of a protein on the SDS-PAGE
gel was detected using tributyrin (TBN) agar plates.
After gel running, the gel was washed sequentially
with 50 mM Tris bu¡er (pH 8.0) containing 1%
(v/v) TX-100 and washed twice with 50 mM Tris
bu¡er. The gel was overlaid on TBN agar plate prepared with agar (1.5%) and tributyrin emulsion (1%
tributyrin, 20 mM NaCl, 1 mM CaCl2 , and 0.5%
(w/v) gum arabic).
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2.3. N-terminal amino acid analysis
The puri¢ed lipase L62 was electrophoretically
transferred to a polyvinylidene di£uoride (PVDF)
membrane (Bio-Rad Lab., Hercules, CA) from
SDS-polyacrylamide gel [14]. The lipase band was
cut out and its amino acid sequence was analyzed
by Edman degradation using an Applied Biosystems
model 476A protein/peptide sequencer (Applied Biosystems Inc.).
2.4. Cloning of the lipase L62 gene
To clone the lipase L62 gene, we made two degenerated primers: primer 1: 5P-CAA(G)TAT(C)AAA(G)AAT(C)AAA(G)TAT(C)CC-3P; primer 2: 5P-GTT(C)TG(ATGC)CC(ATGC)CCCAT(ATGC)CTA(G)TG-3P.
The nucleotide sequence of primer 1 was designed
based on the N-terminal amino acid sequence (Q-YK-N-K-Y-P) of the puri¢ed mature lipase enzyme. In
the case of primer 2, the sequence was designed
based on the highly conserved sequence around the
active site Ser residue. These two primer sets and
S. haemolyticus genomic DNA made a 350-bp PCR
product. This PCR product was used for Southern
hybridization as a probe after labeling with the DIG
DNA Labeling Kit (Boehringer Mannheim, Germany).
The genomic DNA digested with ClaI and EcoRV
showed a hybridization signal at around 4.2 kb. The
corresponding DNA fragment was eluted from the
gel and ligated with the plasmid pBluescript II SK(+)
previously digested with the same enzymes. Escherichia coli XL1 Blue was transformed with this ligation mixture and plated on LB-TBN plates containing 100 Wg Wl31 ampicillin. After overnight
incubation at 37³C, one clear zone-forming colony
was selected.
3. Results
3.1. Puri¢cation of the lipase L62
S. haemolyticus L62 strain secreted as much as
9800 units l31 of lipase to the culture medium. The
lipase was puri¢ed from the culture medium by am-
387
monium sulfate precipitation and three successive
ion exchange column chromatographies. This enzyme's high pI value (9.7), which was obtained using
the Novex Xcell II1 Mini-Cell kit with a pre-cast
IEF gel (pH 3^10), was used e¡ectively in the enzyme puri¢cation process. The lipase passed through
the DEAE column, while most other extracellular
proteins including a 34-kDa protease protein bound
tightly to the DEAE resin. Fig. 1 shows that a 45kDa protein was puri¢ed to homogeneity and after
its renaturation process, it showed a lipolytic activity
on the overlaid TBN agar plate. The speci¢c hydrolytic activity of the puri¢ed lipase was 596 U mg31
and the puri¢cation yield was calculated to be about
42% of the total activity in the culture medium.
The N-terminal 24-amino acid sequence of the 45kDa lipase enzyme was determined to be NH2 -A-TI-K-S-N-Q-Y-K-N-K-Y-P-V-V-L-V-H-G-F-L-G-LV-. Seven amino acid sequences (Q-Y-K-N-K-Y-P)
were used to make degenerated primers in our successive cloning experiment.
Fig. 1. SDS-PAGE (A) and zymogram (B) of S. haemolyticus
L62 lipase. A: The enzyme was electrophoresed on a 12% SDS
polyacrylamide gel. Lane M: the standard proteins ; lane 1: culture supernatant ; lane 2: ammonium sulfate precipitates ; lane 3:
sample after DEAE Sepharose CL-6B chromatography ; lane 4:
sample after CM Sepharose CL-6B chromatography ; and lane 5:
sample after Resource S chromatography. B : The 45-kDa protein
showed lipolytic activity on TBN agar plate.
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Fig. 2. E¡ects of temperature on enzyme activity and stability. A: The enzyme (1 mg ml31 in 20 mM Tris-HCl, pH 7.8) was preincubated
at various temperatures for 30 min in the presence of 10 mM Ca2‡ (E) or 5 mM EDTA (F) and the residual activity was measured by
the pH-stat method at 28³C and pH 8.5. The enzyme was assayed at various temperatures (b). B : The logarithm of the enzyme turnover
rate (k) (s31 ) was plotted against the reciprocal of absolute temperature (T). The values shown are activation energy calculated from the
linear part of the plot.
3.2. Characterization of the lipase L62
The optimum temperature of the lipase L62 was
28³C and its activation energy for the hydrolysis of
olive oil was calculated to be 8.63 kcal mol31 in the
range 4^28³C (Fig. 2). This value is much lower than
those shown by enzymes from other sources: Antarctic bacteria, 12^kcal mol31 , and mesophilic Pseudomonas aeruginosa, 25 kcal mol31 [15,16]. This implies that its catalytic e¤ciency is high in this
Fig. 3. E¡ects of pH on enzyme activity and stability. A: The lipase activity was measured at various pHs by the pH-stat method at 28³C. B: For pH stability test, the enzyme was preincubated at various pH bu¡ers for 24 h at 4³C and the residual
activity was measured by the pH-stat method at 28³C and pH
8.5. E, 0.1 M sodium acetate (pH 4^6); a, 0.1 M potassium
phosphate (pH 6.5^7.5); F, 0.1 M Tris-HCl (pH 7.5^9); O, 0.1
M KCl-glycine-KOH (pH 9^11) ; b, 0.1 M potassium phosphate
(pH 11^12).
temperature range. In fact, the lipase L62 showed
at 4³C as much as 30% of the activity shown by it
at 28³C. In the range over 28³C, its inactivation energy was determined to be 316.4 kcal mol31 .
The lipase L62 showed Ca2‡ -dependent stability at
elevated temperatures. In the presence of Ca2‡ , it is
stable up to 50³C and a drastic decrease in stability
occurred over 55³C, while its thermal stability was
decreased by 10^15³ in the absence of Ca2‡ .
The lipase L62 showed high activity at pH 8.5^9.5
when assayed at 28³C. Although its lipase activity is
somewhat di¡erent depending on the various incubation bu¡ers used, the lipase L62 was fairly stable for
24 h from pH 5 to pH 11 (Fig. 3).
As shown in Fig. 4A, the enzymatic hydrolysis of
triacylglycerols by the lipase L62 depend on the acyl
chain length in the substrate molecules. Tributyrin,
tripropionin, and trimyristin are the preferred substrates whereas changing the length of the acyl
chains dramatically reduces its activity. It showed
about 2-fold higher activity against 1,3-rac-diolein
and a little less activity against 1-rac-monoolein in
comparison with triolein. On the other hand, the
lipase L62 showed the highest activity toward p-nitrophenyl caprylate (C8 ) among the synthetic substrates tested (C2 ^C18 ) (Fig. 4B). In addition, it
showed no activity against phosphatidylcholine
(data not shown), implying that it has no phospholipase activity.
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Fig. 4. Hydrolytic activity of the lipase L62 for various lipids.
A: The hydrolytic activity of the lipase L62 for various chain
lengths of triglycerides, 1,3-rac-diolein (1,3-DO), and 1-rac-monoolein (1-MO), was measured by the pH-stat method using each
substrate emulsion (20 mM) prepared as described in Section 2
for olive oil emulsion. B: Its hydrolytic activity for p-nitrophenyl
esters was measured by the spectrophotometric method. Two different assay methods were used for short chain (C2 ^C12 , E) and
long chain substrates (C12 ^C18 , F) as described in Section 2.
3.3. Cloning and nucleotide sequence of the lipase L62
gene
An E. coli transformant forming a clear halo on
the TBN agar plate was selected as described previously. The recombinant plasmid (pSHL) isolated
from this transformant had about 4.2 kb insert
DNA.
Computer analysis found one major open reading
frame of 2136 bp, which encodes a polypeptide of
711 amino acid residues (Fig. 5). The region upstream of the initiation codon ATG at position 1
contained a putative ribosome binding site and
335 and 310 promoter sequences. A 60-amino
acid signal sequence and a cleavage site between
Ala-60 and Ala-61 were con¢rmed by N-terminal
amino acid analysis of the lipase puri¢ed from
E. coli XL1 Blue (pSHL). In addition, a 259-amino
acid pro-region and a cleavage site between Glu-319
389
and Ala-320 were also determined by N-terminal
amino acid analysis of the 45-kDa mature lipase puri¢ed from the S. haemolyticus strain. Therefore, the
deduced sequence of the mature lipase contains 392
amino acids and corresponds to a molecular mass of
43 841 Da.
In our cloning experiment, E. coli XL1 Blue strain
transformed with the plasmid (pSHL) carrying the
lipase L62 whole gene showed low lipase activity.
This low lipase activity in the recombinant E. coli
strain in comparison with the original staphylococcal
strain (L62) was possibly caused by a low expression
level of this staphylococcal gene in the E. coli system.
In fact, we detected active prolipase protein from
cell-free extract of the transformed E. coli (data not
shown), implying that no processing of the proenzyme occurred in E. coli cells, although the N-terminal signal sequence of the expressed preproenzyme
was correctly removed.
The predicted amino acid sequence of S. haemolyticus lipase was compared with those of other staphylococcal lipases using the Clustal method. The mature part of the lipase L62 has 67%, 53%, 54%, 54%,
and 49% homologies with S. aureus PS54, S. aureus
NCTC8530, S. epidermidis RP62A, S. epidermidis 9
and S. hyicus lipases, respectively. In the case of
preproenzyme, the total sequence of the lipase L62
was shown to have 45%, 37%, 38%, 38%, and 35%
homologies with the same staphylococcal lipases, respectively. Sequence alignment of the lipase L62 with
those staphylococcal lipases showed that Ser-119 in
the mature enzyme is located in the conserved G-XS-X-G sequence around the active site serine residue
(Fig. 6). In addition, Asp-310 and His-352 are
thought to be members of a catalytic triad of this
enzyme together with Ser-119.
4. Discussion
S. haemolyticus L62 strain produced and secreted
as much as 9800 U l31 of a 45-kDa lipase to the
growth medium. The amount of the lipase L62 was
calculated to be about 14% of the total extracellular
proteins on the basis of the speci¢c activity (596 U
mg31 ). But from the protein band on the gel presented in Fig. 1, it looks as much as 40% of the total
protein (Fig. 1A, lane 2). This discrepancy can be
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explained from two postulations: that the lipase L62
was secreted as a complex with some compound,
which complex has been already reported from several lipases [17] and that this `lipase complex' has
much lower lipolytic activity in comparison with
the puri¢ed enzyme. This lipase complex was evidenced from our two experimental results. First,
the lipase L62 in culture supernatant was resistant
to the attack of the endogenous 34-kDa protease,
whereas the puri¢ed enzyme was rapidly degraded
by it. Second, the total lipase activity was rather
increased after ammonium sulfate precipitation in
the puri¢cation process (data not shown).
The cloned lipase gene suggests that the staphylococcal lipase is produced in an 80-kDa preproenzyme
form, secreted, and processed rapidly to the 45-kDa
Fig. 5. Nucleotide sequence of the L62 lipase gene and its deduced amino acid sequence. The numbering of nucleotides starts at the 5P
end of the lipase gene and that of amino acids at the N-terminus of the preproenzyme. The putative 335, 310, ribosomal binding site
(rbs), and stop codon (*) are shown. The N-terminal amino acid sequence from the puri¢ed proenzyme and mature enzyme are underlined. The sequence has been submitted to GenBank under accession number AF096928.
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391
Fig. 6. Primary structure alignment of the mature lipases from S. haemolyticus (a), S. aureus PS54 (b), S. aureus NCTC8530 (c), S. epidermidis (d) and S. hyicus (e). In this alignment, the numbering starts at the N-terminal residue of the mature sequences. Amino acids conserved in all ¢ve lipases (*) and the putative active site residues (2) are indicated. Arrows indicate the Ca2‡ binding sites (u) and the Ser356 residue (v) in SHL.
mature enzyme in this staphylococcal strain. In our
experiment, neither prepro- nor proenzyme bands
could be detected by Coomassie staining or activity
staining of the SDS-PAGE gel.
The optimum temperature for the lipase L62 was
determined to be 28³C, however, it showed as much
as 30% activity at 4³C (Fig. 2). This high activity at
low temperature makes the enzyme applicable as an
additive to detergents used at low temperatures and
as a biocatalyst for biotransformation of labile compounds at cold temperatures.
Sequence alignment using the Clustal method
showed the lipase L62 has high homologies (49^
67%) with other staphylococcal lipases, whereas it
has less than 30% homology with all other bacterial
lipases. This result shows that on the basis of sequence homology, the lipase L62 together with other
staphylococcal lipases seems to make a separate li-
pase group. Although SAL and SHL have high homology (50^51%) in their amino acid sequences,
their enzymatic properties such as substrate speci¢city are very di¡erent. This discrepancy in substrate
speci¢city between these two homologous enzymes
has not been explained clearly, for no X-ray crystal
structure for the staphylococcal lipase has been elucidated yet. However, Nikoleit and co-workers
showed that the C-terminal 146 amino acids of
SHL are required for its phospholipase activity [4].
After that, Kampen and co-workers showed that
three regions in the C-terminal domain of SHL
were responsible for phospholipase activity and a
region of residues 180^250 in SHL for its broad
chain length selectivity [10]. Recently, they suggested
that the Ser-356 residue plays an important role in
determining phospholipase activity in SHL [18]. In
the case of the lipase L62, it has no phospholipase
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activity and shows relatively high hydrolytic activity
for short chain length triglycerides such as tributyrin
and tripropionin (Fig. 4) like SAL. This similarity in
substrate speci¢city between the lipase L62 and SAL
correlates well with their relatively high homology in
total sequences (67%) or C-terminal part (146 amino
acids) sequence (56%). In particular, the lipase L62
and SAL have Leu and Val residues, respectively, in
the position matching the Ser-356 of SHL.
On the other hand, Simons and co-authors showed
that Asp-354 and 357 could be Ca2‡ binding sites in
SHL by site-directed mutagenesis in combination
with isothermal titration calorimetry and they suggested that the calcium binding to the residues is
important for its structural stabilization at elevated
temperatures [19]. The lipase L62 was also stabilized
at elevated temperatures by calcium binding (Fig. 2).
Sequence alignment of the lipase L62 with SHL suggested that residues Asp-351 and Asp-354 are calcium binding sites in the lipase L62. But to determine
de¢nitely the motifs involved in calcium binding or
substrate binding in the lipase L62 enzyme, more
detailed biochemical studies in combination with
X-ray crystallography are needed.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
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