Purification and Characterization of A Monoacylglycerol Lipase From The Moderately Thermophilic Bacillus Sp. H-257

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J. Biochem.

127, 419-425 (2000)

Purification and Characterization of a Monoacylglycerol Lipase from


the Moderately Thermophilic Bacillus sp. H-257
Shigeyuki Imamura1 and Shiro Kitaura2
Diagnostic Research and Development, Diagnostic Division, Asahi Chemical Industry Co., Ltd., 632-1 Mifuku,
Ohito, Shizuoka 410-2321

Received October 22, 1999; accepted December 13,1999

A thermostable monoacylglycerol lipase [MGLP, EC 3.1.1.23] was purified for the first
time from a cell-free extract of the moderately thermophilic Bacillus sp. H-257. The

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enzyme was purified 3,028-fold to homogeneity by chromatography using Octyl-
Sepharose CL-4B, Q-Sepharose FT, and Superose 12 columns. The molecular mass of the
MGLP was estimated to be 25 kDa by gel nitration and 24 kDa by SDS-PAGE, suggesting
a monomeric protein. The isoelectric point was determined to be 4.66 by isoelectric
focusing. The MGLP retained its full activity upon incubation at 60°C for 10 min (pH
7.3), and was stable at pH 7-10. The optimal temperature for activity at pH 7.5 was 75°C,
and the maximum activity was observed from pH 6-8. This enzyme hydrolyzes monoa-
cylglycerols, with the highest activity occurring with 1-monolauroylglycerol. Di- and tri-
acylglycerols, on the other hand, are essentially inert as substrates for the enzyme. The
Km values for the hydrolysis of 1-monolauroylglycerol, 1-monooleoylglycerol, and 2-
monooleoylglycerol were determined to be 140, 83 and 59 JAM, respectively. The enzyme
was not inhibited by cholate, but was slightly inhibited by Triton X-100 and deoxycho-
late. The amino acid sequence of the N-terminal region of the enzyme (16 residues) was
also determined.

Key words: moderately thermophilic bacteria, monoacylglycerol lipase, N-terminal


amino acid sequence, purification, thermostable enzyme.

Lipase [triacylglycerol lipase, EC 3.1.1.3] hydrolyzes tri- In recent years, there has been a great demand for ther-
acylglycerol to diacylglycerol, monoacylglycerol, fatty acid, mostable enzymes in industrial fields. Thus, thermostable
and glycerol. This enzyme, which is widely distributed in lipases from various sources have been purified and charac-
animals, plants, and microorganisms, is important and use- terized (17-19). Also, proteins from thermophilic organisms
ful in various industrial fields as a biocatalyst. A lipase that have proved to be more useful for biotechnological applica-
hydrolyzes partial acylglycerol, mono-, and diacylglycerols tions than similar proteins from methophiles due to their
has been purified and characterized (1-10). Yamaguchi et stability.
al. (11) reported an enzyme from Penicillium camenbertii We tried to screen a thermostable MGLP from bacterial
that hydrolyzes mono- and diacylglycerols but not triacyl- strains, and found it in the cell extract of the moderately
glycerols. Ikeda et al. (12) reported a rat liver microsomal thermophilic Bacillus sp. H-257 isolated from soil. Li this
lipase that maximally hydrolyzes long chain monoacylglyc- study, we report the purification and enzymatic properties
erols, and 1(3)- and 2-monoacylglycerols, but shows a little of this MGLP.
hydrolytic activity for short chain triacylglycerols, and no
hydrolytic activity for long chain triacylglycerols. Until now MATERIALS AND METHODS
only limited information on a specific monoacylglycerol-
hydrolyzing enzyme, monoacylglycerol lipase [MGLP, EC Materials—Pharmamedia (defatted cotton seed meal)
3.1.1.23], has been available. MGLPs from mouse (13) and was a product of Traders Oil Mill (Fort Worth, TX, USA).
rat (14) adipocytes, and from rabbit aorta (15) and human Peptone was from Difco Laboratories (Detroit, MI, USA).
erythrocytes (16) have been reported. However, no MGLP Silicone KM-72 (anti-foaming agent) was a product of Shin-
from bacteria has yet been described. Etsu Chemical Industry (Tokyo). Adekatol SO-120 was a
product of Asahi-denka Kogyo (Tokyo). 1-Monoacetylglyc-
1
To whom correspondence should be addressed. Fax: +81-558-76- erol and 1-monobutyrylglycerol were products of Kanto
7149, Tel: +81-558-76-7050, E-mail: a7086288@ut.asahi-kasei.co.jp Chemical (Tokyo). 1-Monocaprylglycerol and 1-monostearo-
2
Present address: Clinical Development Center, Asahi Chemical ylglycerol were products of Larodan Fine Chemicals AB
Industry Co., Ltd., 9-1 Kanda Mitoshirocho, Chiyoda-ku, Tokyo 101- (Malmo, Sweden). 1-Monolauroylglycerol, 1-monomirystoyl-
8481. glycerol, 1-palmitoylglycerol, 1-monolinoleoylglycerol, and
Abbreviations: B. sp., Bacillus sp.; GK, glycerol kinase; GPO, glyc- 2-monooleoylglycerol were from Serdary Research Labora-
erol-3-phosphate oxidase; MGLP, monoacylglycerol lipase; TOOS,
iV-ethyl-Af-(2-hydroxy-3-sulfopropyl)-m-toluidine. tories (Toronto, Ontario, Canada). 1-Monooleylglycerol, 1-
monolinolenoylglycerol, and 1,2-dilinoleoylglycerol were
© 2000 by The Japanese Biochemical Society. prducts of Nu-Chek-Prep (Elysian, MN, USA). Para-nitro-

Vol. 127, No. 3,2000 419


420 S. Imamura and S. Kitaura

phenyl acetate, p-nitrophenyl butylate, p-nitrophenyl caply- ugation (15,000 xg for 10 min) and dissolved in 130 ml of 10
late, p-nitrophenyl laurate, and p-nitrophenyl palmitate mM potassium phosphate buffer (pH 7.0) containing 10%
were products of Sigma Chemical (St. Louis, MO, USA). ammonium sulfate. The undissolved matter was removed
Glycerol kinase (GK) and glycerol-3-phosphate oxidase by centrifugation (15,000 xg for 10 min), and the superna-
(GPO) were products of Asahi Chemical Industry (Tokyo). tant obtained was used for the next purification procedure.
Peroxidase was from Sigma Chemical (St. Louis, MO, Step 3. Octyl-Sepharose CL-4B column chromatography:
USA). TOOS [A^-ethyl-N-(2-hydroxy-3-sulfopropyl)-/n-tolui- The enzyme solution (125 ml) was applied to an Octyl-
dine] was obtained from Dojindo Laboratories (Kumamoto). Sepharose CL-4B column (2.5 cm x 21 cm) equilibrated
Octyl-Sepharose CL-4B, Q-Sepharose FF, Superose 12 col- with 10 mM potassium phosphate buffer (pH 7.0) contain-
umns, and Ampholine were from Pharmacia (Uppsala, ing 10% ammonium sulfate. Washing of the column was
Sweden). The TSK gel G3000SWXL column was from Tohso carried out stepwise as follows: 100 ml of 10 mM potassium
(Tokyo). Molecular weight marker proteins for gel filtration phosphate buffer (pH 7.0) containing 10% ammonium sul-
and yeast extract were from Oriental Yeast (Tokyo). All fate, 100 ml of the same buffer containing 5% ammonium
other chemicals were of analytical grade and commercially sulfate, 100 ml of the same buffer containing 2.5% ammo-

Downloaded from http://jb.oxfordjournals.org at Addis Ababa University Libraries on May 31, 2010
available. nium sulfate, 100 ml of the same buffer containing 1.0%
Microorganism and Growth Condition—B. sp. H-257 ammonium sulfate, 200 ml of 10 mM potassium phosphate
originally isolated from soil was used as a source of MGLP. buffer (pH 7.0). The enzyme was eluted with 10 mM potas-
A medium of the following composition (pH 7.0) was used sium phosphate buffer (pH 7.0) containing 0.5% Adekatol
for the growth of the cells: glucose; 0.2% (w/v); Pharmame- SO-120 at a flow rate of 100 ml per h. The active fractions
dia; 0.5%; peptone, 1.0%; yeast extract, 0.5%; NaCl, 0.3%; were collected.
KELPC^, 0.1%; MgSO4.7H2O, 0.05%; and Silicone KM-72, Step 4. Q-Sepharose FF column chromatography: The
0.05%. The bacterium was cultivated at 50°C for 12 h in a active fractions (160 ml) were applied to a Q-Sepharose FF
30-liter jar fermentor under aeration (20 liters per min). column (2.5 cm x 10 cm) equilibrated with 10 mM potas-
Enzyme Assay and Protein Determination—Lipase activ- sium phosphate buffer (pH 7.0). After the column was
ity was determined spectrophotometrically as follows. The washed with the same buffer (200 ml), the enzyme was
reaction mixture, containing 100 ul of 0.2 M PIPES-NaOH eluted with a linear gradient of KC1 from 0 to 0.3 M in a
(pH 7.3), 50 |il of 0.3% 4-aminoantipyrine, 50 ul of 0.2% total volume of 600 ml. Fractions of 20 ml were collected at
TOOS, 50 ul of 45 U/ml peroxidase, 25 ul of 20 mM MgCL,, a flow rate of 100 ml per h.
25 ul of 20 mM ATP, 10 ul of 25 U/ml GK, 15 ul of 1,000 U/ Step 5. Superose 12 column chromatography: After dialy-
ml GPO and 75 ul of fL.0, and 50 ul of substrate solution sis of the enzyme solution against 20 volumes of buffer, the
(10 mM monolauroylglycerol containing 0.5% Triton X- enzyme solution (1 ml) concentrated by Centricon (Amicon)
100), was kept at 37°C, and the reaction was started by was applied to a Superose 12 column (2.0 cm x 52 cm),
adding enzyme solution (50 ul). After incubation at 37°C for equilibrated with 10 mM potassium phosphate buffer (pH
10 min, the reaction was stopped by adding 2.5 ml of 0.5% 7.0) containing 0.5 M KC1 at a flow rate of 1 ml per h. Frac-
SDS solution. The glycerol generated was measured by the tions of 1 ml were collected and the active fractions were
absorbance at 550 nm. One unit (U) of enzyme activity was dialyzed.
defined as the amount of enzyme that liberates 1 umol of Determination of Molecular Mass—The molecular mass
glycerol per min at 37°C under the conditions specified of the native enzyme was estimated by gel filtration on a
above. Specific activity was expressed as U/mg protein. Pro- TSK gel G3000SWXL column (30 cm) using 50 mM sodium
tein concentration was determined with a Bio-Rad protein phosphate buffer (pH 7.5) containing 0.2 M Na^O,,. The
assay kit (Richmond, CA, USA) using BSA as a standard following standard proteins were used to create a calibra-
protein. tion curve: glutamate dehydrogenase (Mr 290 kDa), lactate
Purification of MGLP—All operations were performed at dehydrogenase (142 kDa), enolase (67 kDa), adenylate
room temperature and potassium phosphate buffer was kinase (32 kDa), and cytochrome c (12.4 kDa). Molecular
used as a standard buffer throughout the purification. mass of the subunit was determined by SDS-PAGE using a
Step 1. Preparation of crude extract: B. sp. H-257 cells 12.5% polyacrylamide gel. The marker proteins used were
obtained from a 20-liter medium culture were suspended in as follows: bovine serum albumin (67 kDa), ovalbumin (45
2 liters of 10 mM potassium phosphate buffer (pH 7.0) con- kDa), bovine pancreas a cymotrypsinogen A (25.7 kDa),
taining 0.2% (w/v) lysozyme and 0.2% Triton X-100, then cytochrome c (12.4 kDa). Proteins were visualized by Coo-
incubated at 37°C for 2 h. After centrifugation (15,000 xg massie Brilliant Blue staining.
for 20 min), the supernatant (1,960 ml) was concentrated Substrate Specificity—Substrate specificity toward differ-
by ultrafiltration to 550 ml. This solution was used as a ent partial acylglycerols (C2-C18 acyl groups) at 1 mM was
crude extract for the further purification procedures. determined under the standard assay conditions described
Step 2. Acetone fractionation and precipitation: Cold ace- above. Substrate specificity toward different p-nitrophenyl
tone (275 ml) was added to the crude extract, and the pre- esters (C2-C16 acyl groups) was determined as follows. The
cipitate formed was removed by centrifugation. Then cold reaction mixture, containing 100 ul of 0.2 M PIPES-NaOH
acetone (671 ml) was added again to the supernatant solu- (pH 7.3), 50 ul of substrate solution (1 mM p-nitrophenyl
tion (746 ml), and the precipitate formed was collected by ester containing 1% Triton X-100), and 350 ul of H,O, was
centrifugation (15,000 xg for 15 min). The precipitate was kept at 37°C, and the reaction was started by adding en-
dissolved in 250 ml of 10 mM potassium phosphate buffer zyme solution (25 ul). After incubation at 37°C for 10 min,
(pH 7.0). The supernatant (236 ml) was obtained by centrif- the reaction was stopped by adding 1 ml of 2% SDS solu-
ugation (15,000 xg for 10 min), and cold acetone (472 ml) tion. The absorbance at 420 nm was determined. The activ-
was added. The precipitate formed was collected by centrif- ity of MGLP toward different triacylglycerols was deter-

J. Biochem.
Monoacylglycerol Lipase from Moderately Thermophilic Bacillus sp. 421

mined by alkaline titration according to a method described amino acid sequence of the N-terminal region of the puri-
previously (20). fied MGLP was identified by automated Edman degrada-
Measurement of Isoelectric Point (pi)—Isoelectric focusing tion in a gas-phase protein sequencer (Applied Biosystems,
was carried out at 25CC for 40 h with Ampholine carrier USA).
ampholytes giving a pH gradient of 3.5 to 10 in a 110-ml
electrofocusing column. RESULTS AND DISCUSSION
Effects of pH and Temperature on Enzyme Activity—The
pH effect on activity was identified by measuring the We isolated several MGLP-producing bacterial strains from
enzyme activity at various pH in the range of 4—9.5, using soil samples in Kagoshima prefecture. Among them, strain
1-monolauroylglycerol (1 mM) as a substrate. The tempera- H-257 was selected for its MGLP activity, which hydrolyzes
ture effect on activity was identified by measuring the rela- monoacylglycerols but not di- and triacylglycerols. On the
tive activity at specified temperatures in the range of 30°C basis of strain characteristics, strain H-257 was identified
-85°C. as a moderately thermophilic bacterium of the genus Bacil-

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Effects ofpH and Temperature on Enzyme Stability—The lus.
pH effect on stability was evaluated by measuring residual Culture Conditions for the High Production of MGLP—B.
activity after incubation of the enzyme at 60°C for 10 min sp. H-257 was cultured in medium containing different car-
at various pH, using the following buffers: DMG-NaOH bon and nitrogen sources to find the conditions leading to
(pH 4.5-7), PIPES-NaOH (pH 6.5-7), Tris-HCl (pH 7-9), the highest production of the enzyme. The highest produc-
Glycine-NaOH (pH 9-10). For the determination of the tion was observed in the cell extract when the strain was
effect of temperature on stability, residual activities were cultured for 12 h under the conditions and in the medium
measured after incubation of the enzyme at various tem- described in "MATERIALS AND METHODS".
peratures for 10 min. Most microbial lipases are extracellular, and secreted
Analysis of N-Terminal Amino Acid Sequence—The through the external membrane into the culture medium
(21). Lipases produced by bacteria of the genus Bacillus
(22-24), Pseudomonas (25), and Candida (26) are well
100

o
s
Oil

o
CO
CM

Fraction No.
Fig. 1. Column chromatography on Octyl-Sepharose CL-4B. 10 20 40
The enzyme solution (125 ml) after acetone fractionation (33.3— Fraction No.
77.7%) was applied to an Octyl-Sepharose CL-4B column (2.5 cm x Fig. 2. Column chromatography on Q-Sepharose FF. The en-
21 cm) previously equilibrated with 10 mM phosphate buffer (pH zyme solution (160 ml) obtained from the Octyl-Sepharose CL-4B
7.0) containing 10% ammonium sulfate. The column was washed column was applied to a Q-Sepharose FF column (2.5 cm x 10 cm)
with 100 ml of the same buffer, 100 ml of 5% ammonium sulfate, 100 previously equilibrated with 10 mM phosphate buffer (pH 7.0). The
ml of 2.5% ammonium sulfate, 1% ammonium sulfate, and 200 ml of column was washed with 200 ml of the same buffer and eluted with
10 mM phosphate buffer (pH 7.0) and then eluted with 10 mM phos- 600 ml of a linear gradient of 0 to 0.3 M KC1 containing 10 mM
phate buffer containing 0.5% Adekatol SO-120 at a flow rate of phosphate buffer (pH 7.0) at a flow rate of 100 ml per h, and frac-
about 100 ml per h. Fractions of 20 ml were collected. All procedures tions of 20 ml were collected. All procedures were carried out at
were carried out at 25°C. Other experimental conditions are de- 25°C. Other experimental conditions are described in the text. •,
scribed in the text, o, absorbance at 280 ran; •, MGLP activity. MGLP activity; o, absorbance at 280 nm.

TABLE I. Summary of the purification of MGLP from Bacillus sp. H-257.


Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification (fold) Yield (%)
Cell free extract 82,288 3,197 0.04 1 100
Acetone fractionation 41,412 2,888 0.07 1.7 90
Acetone precipitation 1st 20,691 1,736 0.08 2.0 54
2nd 13,728 1,244 0.09 2.3 39
Octyl-Sepharose CL-4B 271 947 3.5 88 30
Q-Sepharose 8.8 819 93.0 2,325 26
Superose 12 5.3 642 121.1 3,028 20

Vol. 127, No. 3,2000


422 S. Imamura and S. Kitaura

known to be secreted extracellularly. However, the novel The enzyme appeared as a single band on SDS-PAGE at a
MGLP produced by B. sp. H-257 is not secreted extracellu- migration distance corresponding to a molecular mass of
larly and accumulates in cells in an active form, making approximately 24 kDa (Fig. 4). This indicates that MGLP is
MGLP a unique enzyme. catalytically active in a monomeric form. The molecular
Purification of MGLP—MGLP from B. sp. H-257 was masses of MGLP from mouse/rat adipocytes and human
purified 3,028-fold in 5 steps to a measured final specific erythrocytes were approximately 33 kDa and 60 + 2 kDa,
activity of 121.1 U/mg-protein with a yield of 20% (Table I). respectively (13, 14, 16). These were also monomeric. The
The specific activity of the enzyme was increased 2.3-fold apparent pi of MGLP, as determined by an electrofocusing
by acetone precipitation. The next step, hydrophobic chro- column, was around 4.66.
matography on Octyl-Sepharose CL-4B, was effective for Effects of pH and Temperature on Enzyme Activity—The
the purification of the enzyme with an increase in the spe- effects of pH and temperature on MGLP activity were
cific activity of about 88-fold. Anion exchange chromatogra- examined. When assayed at various pH at 37°C, MGLP
phy on Q-Sepharose was successfully employed to increase
the specific activity 2,325-fold with a recovery greater than

Downloaded from http://jb.oxfordjournals.org at Addis Ababa University Libraries on May 31, 2010
25%. Elution profiles from the Octyl-Sepharose CL-4B, Q- 120
Sepharose FF and Superose 12 columns are shown in Figs.
1-3.
Molecular Mass and pi—The molecular mass of purified
MGLP was determined to be about 25 kDa by gel nitration.

Fig. 5. Effect of pH on the activity of monoacylglycerol li-


pase. The enzyme activity at various pH values was assayed in the
reaction mixture described in the text. Acetate buffer was used for
pH 4 to 6 (o); phosphate buffer for pH 6 to 8 (•); Tris-HCl buffer for
CD
pH 7 to 9 (a); glycine-NaOH buffer for pH 9 to 10 (•). The enzyme
source was the "Superose 12fraction"of Table I.

50 60 70
Fraction No.
Fig. 3. Column chromatography on Superose 12. The enzyme
solution (1 ml) obtained from the Q-Sepharose FF column was ap-
plied to a Superose 12 column (2.0 cm x 52 cm) at 25°C previously
equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.5M
KC1. Fractions of lml were collected at a flow rate of 1 ml per min.
Other experimental conditions are described in the text. •, MGLP
activity; O, absorbance at 280 nm.

1 2 kDa

Temperature CC)
67 Fig. 6. Effect of reaction temperature on monoacylglycerol
lipase activity. The reaction mixture, containing 100 ul of 0.2M
PIPES-NaOH (pH 7.3), 50 ul of 10 mM monolauroylglycerol contain-
45 ing 0.5% Triton X-100, and 300 nl of HjO was kept at various tem-
perature described in Fig. 6. The reaction was started by adding 50
• 25.7 ul of enzyme solution, and stopped by adding 100 fxl of 2 M HC1.
After incubation at 37°C for 10 min, 100 JJ.1 of 2 M NaOH was added
to the reaction mixture. The glycerol generated was determined col-
orimetrically by adding 500 ul of enzyme reagent containing 100 ul
12.4 of 0.3% 4-aminoantipyrine, 100 ul of 0.2% TOOS, 100 ul of 45U/ml
peroxidase, 50 ul of 20 mM MgCL,, 50 ul of 20 mM ATP, 20 ul of 25U/
Fig. 4. SDS-PAGE of MGLP. Electrophoresis was carried out as de- ml glycerol kinase, 30 ul of 1,000 U/ml glycerol-3-phosphate oxidase,
scribed under "MATERIALS AND METHODS". After electrophore- and 50 ul of JL.O. After incubation at 37°C for 10 min, 2 ml of 0.5%
sis, the gel was stained with Coomasie Brilliant Blue. Lane 1, SDS solution was added, and the absorbance at 550 nm was mea-
Bacillus sp. H-257 MGLP; lane 2, molecular marker proteins. sured.

J. Biochem.
Monoacylglycerol Lipase from Moderately Thermophilic Bacillus sp. 423

showed high activity around pH 6-8 (Fig. 5). MGLP was no measurable activity was detected for 1,2-diacylglycerols,
most active at 75°C, when assayed for 10 min at pH 7.5 diacylglycerol from egg yolk phosphatidylcholine, on tria-
(Fig. 6). cylglycerols.
Effects of pH and Temperature on Enzyme Stability— The substrate specificity toward p-nitrophenyl esters (1
From pH stability tests using various buffers, it was found mM) was also determined. The MGLP showed the highest
that the MGLP is stable from pH 7 to 10 after incubation activity toward p-nitrophenyl butylate (C4 acyl group)
at 70°C for 10 min, as shown in Fig. 7. Up to 60°C, the among the substrates examined, which is 23.9% of the
MGLP is stable for 10 min, but above 60°C it is inactivated activity toward 1-monolauroylglycerol. Not only monoacylg-
(Fig. 8). lycerols (alcoholic hydroxyl esters) but also p-nitrophenyl
Catalytic Properties—The MGLP showed the highest esters (phenolic hydroxyl esters) were substrates for MGLP.
activity toward 1-monolauroylglycerol (C12 acyl group) MGLP had higher activity toward 1-monolauroylglycerol
among the substrates examined (C2-C18 acyl groups), as (C12 acyl group) among partial acylglycerols than the activ-
shown in Table II. When the acyl chain length of the sub- ity toward p-nitrophenyl butylate (C4 acyl group) among p-

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strate was increased above C12 or decreased below C12, nitrophenyl esters. The enzyme also showed activity toward
there was a steep decrease in enzyme activity. It has been p-nitrophenyl laurate (C12 acyl group) and p-nitrophenyl
reported that the MGLP from rat adipocytes (14) and rab- palmitate (C16 acyl group), suggesting that it has different
bit aorta (75) hydrolyze 1(3)- and 2-monooleoylglycerols substrate specificity from esterases.
(C181) similarly. Thus, we examined the positional specific- An assay method for lipase activity using diacylglycerol
ity of MGLP for monooleoylglycerols. The enzyme had 1.25 as a substrate is known to be useful (27). This method is
times higher activity toward 2-monooleoylglycerol in com- based on the measurement of monoacylglycerol, which is
parison with l(3)-monooleoylglycerol. On the other hand, liberated from diacylglycerol. For this purpose, a specific
monoacylglycerol-hydrolyzing enzyme is essential and a
small Km for monoacylglycerol is preferable. Therefore, we
120 examined the Kms of the enzyme described here. Initial
velocity analysis showed that the enzyme exhibits Michae-
lis-Menten type kinetic patterns. Kms for the hydrolysis of
1-monolauroylglycerol, 1-monooleoylglycerol, and 2-mono-
oleoylglycerol were determined to be 140, 83, and 59 uM,

TABLE II. Substrate specificity of MGLP toward mono-, di-,


triacylglycerols.
Substrate Relative activity (%)
1-Monoacetylglycerol (C2.0) 10.5
1-Monobutyroylglycerol (C40) 47
1-Monocapryloylglycerol (C8;0) 94
1-Monolauroylglycerol (C120) 100
pH
1-Monomyristoylglycerol (CH;0) 84.5
Fig. 7. Effect of pH on the stability of monoacylglycerol 1-Monoparmitoylglycerol (C16:0) 63.5
lipase. The enzyme solution (10 U/ml) in 10 mM buffer was incu- 1-Monostearoylglycerol (C18:0) 45.5
bated for 60 min at 45°C. Dimethylglutarate-NaOH buffer was used 1-Monooleoylglycerol (C181) 57
for pH 4 to 6 (o); PIPES-NaOH buffer for pH 6 to 8 (•); Tris-HCl 1-Monolinoleoylglycerol (C182) 75
buffer for pH 7 to 9 (D); glycine-NaOH buffer for pH 9 to 11 (•). The 1-Monolinolenoylglycerol (C18;3) 81.5
enzyme source was the "Superose 12 fraction" in Table I. 2-Monooleoylglycerol (C18.,) 71.5
1,2-Dilinoleoylglycerol (C18:2) 0
Triacetylglycerol (C2:0) 0
Tributyroylglycerol (C40) 0
Trioleoylglycerol (C18:1) 0
100
p-Nitrophenyl acetate (C20) 9.3
p-Nitrophenyl butylate (Q,.o) 23.9
* 80
p-Nitrophenyl caplylate (C8;0) 12.7
p-Nitrophenyl laurate (C12:0) 6.2
- 60 p-Nitrophenyl palmitate (C16:0) 1.9

40
TABLE III. Effect of detergents on MGLP activity.
20 Detergent Concentration Relative activity I
None 100
Triton X-100 0.05 % 100.2
20 40 60 80 0.25 % 90.3
Temperature ( "C ) 0.65 % 89.7
Fig. 8. Effect of temperature on the stability of monoacylglyc- 1.05 % 89.5
erol lipase. The enzyme solution (10 U/ml) in 10 mM PIPES-NaOH Cholate lmM 97.5
(pH 7.5) buffer was incubated for 10 min at various temperatures. 5mM 99.5
The residual activity was assayed in the reaction mixture described Deoxycholate lmM 94.8
in the text. 5mM 83.1

Vol. 127, No. 3, 2000


424 S. Imamura and S. Kitaura

respectively, suggesting the enzyme has a high affinity for Characterization of mono-, di-, and triacylglycerol lipase activi-
monoacylglycerols. ties in the isolated rat heart. Biochim. Biophys. Ada 875, 76-86
Pancreatic lipase is well known to be strongly activated 8. Keough, D.T., de Jersey, J., and Zerner, B. (1985) The relation-
ship between the carboxylesterase and monoacylglycerol lipase
by deoxycholate, and diacylglycerol is solubilized with non- activities of chicken liver microsomes. Biochim. Biophys. Ada
ionic detergents or deoxycholate. Therefore, the effects of 829, 164-172
detergents on enzyme activity were also determined. As 9. Hee-Cheong, M., Fletcher, T., Kryski, S.K., and Severson, D.L.
shown in Table III, the enzyme was not inhibited by etio- (1985) Diacylglycerol lipase and kinase activities in rat brain
late, but was slightly inhibited by Triton X-100 (1.05%) and microvessels. Biochim. Biophys. Ada 833, 59-68
deoxycholate (5 mM). 10. Farooqui, A.A., Taylor, W.A., and Horrocks, L.A. (1984) Separa-
tion of bovine brain mono- and diacylglycerol lipases by heparin
Many lipases are known to be activated by calcium ions. sepharose affinity chromatography. Biochem. Biophys. Res.
We examined the influence of mono- and divalent ions (10 Commun. 122, 1241-1246
mM) in addition to calcium ions on MGLP activity. None of 11. Yamaguchi, S., Takeuchi, K., Mase, T., and Matsuura, A. (1997)
the following compounds showed any appreciable influence Efficient expression of mono- and diacylglycerol lipase gene
on enzyme activity: NaCl, KC1, LiCl, MgCl2, MnCLj, and from Penicillium camembertii U-150 in Aspergillus oryzae

Downloaded from http://jb.oxfordjournals.org at Addis Ababa University Libraries on May 31, 2010
CaCl2 (data not shown). under the control of its own promoter. Biosci. Biotechnol. Bio-
chem. 61,800-805
The results described above suggest that the enzyme is
12. Ikeda, Y., Okamura, K., and Fujii, S. (1977) Purification and
very useful for application in the assay method described characterization of rat liver microsomal monoacylglycerol
above. lipase in comparison to the other esterases. Biochim. Biophys.
Analysis of the N-Terminal Amino Acid Sequence—The Ada 488,128-139
amino acid sequence of the N-terminal region of the en- 13. Karlsson, M., Contreras, J.A., Hellman, U., Tornqvist, H., and
zyme was determined as follows: Ser-Glu-Gln-Tyr-Pro-Val- Holm, C. (1997) cDNA cloning, tissue distribution, and identifi-
Leu-Ser-Gly-Ala-Glu-Pro-Phe-Tyr-Ala-Glu- (16 residues). cation of the catalytic triad of monoglyceride lipase. Evolution-
This sequence was compared with the sequences deposited ary relationship to esterases, lysophosphospholipases, and
haloperoxidases. J. Biol. Chem. 272, 27218-27223
in the DDBJ database and with three other Bacillus lipase 14. Tornqvist, H. and Belfrage, P. (1976) Purification and some pro-
sequences: B. stearothermophilus (19), B. thermocatenula- perties of a monoacylglycerol-hydrolyzing enzyme of rat adi-
tus (28), and B. subtilis (29). However, no significant homol- pose tissue. J. Biol. Chem. 251, 813-819
ogy to other sequences was found. 15. Hee-Cheong, M. and Severson, D.L. (1987) Properties of monoa-
In this study, we screened a thermostable MGLP from cylglycerol lipase in rabbit aorta. Lipids 22, 999-1004
the moderately thermophilic B. sp. H-257, established a 16. Somma-Delpero, C, Valette, A., Lepetit-Thevenin, J., Nobili, O.,
Boyer, J., and Verine, A. (1995) Purification and properties of a
purification method for the enzyme, and showed its charac- monoacylglycerol lipase in human erythrocytes. Biochem. J.
teristics. To our knowledge, this is the first description of a 312, 519-525
specific monoacylglycerol-hydrolyzing enzyme from a mod- 17. Sugihara, A., Ueshima, M., Shimada, Y, Tsunasawa, S., and
erately thermophilic bacterium. This MGLP accumulates Tominaga, Y. (1992) Purification and characterization of a novel
intracellularly in an active form, and hydrolyzes monoa- thermostable lipase from Pseudomonas cepacia. J. Biochem.
cylglycerol, but not di- and triacylglycerols to an apprecia- 112,598-603
ble extent. It is thermostable up to 60°C, suggesting its 18. Sugihara, A., Tani, T., and Tominaga, Y (1991) Purification and
characterization of a novel thermostable lipase from Bacillus
usefulness for analytical applications such as the measure- sp. J. Biochem. 109, 211-216
ment of serum lipase levels and the analysis of monoacyl- 19. Kim, H.K., Park, S.Y., Lee, J.K., and Oh, T.K. (1998) Gene clon-
glycerols in food. However, the production of MGLP by B. ing and characterization of thermostable lipase from Bacillus
sp. H-257 is low (about 8.3 ng/ml). To increase production stearothermophilus LI. Biosci. Biotechnol. Biochem. 62, 66-71
efficiency, molecular cloning and expression of the struc- 20. Horiuti, Y, Koga, H., and Gocho, S. (1976) Effective method for
tural gene will be necessary. activity assay of lipase from Chromobacterium viscosum. J. Bio-
chem. 80,367-370
21. Taipa, M.A., Aires-Barros, M.R., and Cabral, J.M.S. (1992)
REFERENCES Purification of lipases. J. Biotechnol. 26, 111-142
22. Kim, H.K., Sung, M.H., Kim, H.M., and Oh, T.K. (1994) Occur-
1. Moriyama, T., Urade, R., and Kito, M. (1999) Purification and rence of thermostable lipase in thermophilic Bacillus sp. strain
characterization of diacylglycerol lipase from human platelets. 398. Biosci. Biotechnol. Biochem. 58, 961-962
J. Biochem. 125, 1077-1085 23. Lesuisse, E., Schanck, K., and Colson, C. (1993) Purification
2. Lee, M.W. and Severson, D.L. (1994) Partial purification of a and preliminary characterization of the extracellular lipase of
diacylglycerol lipase from bovine aorta. Biochem. J. 298, 213- Bacillus subtilis 168, an extremely basic pH-tolerant enzyme.
219 Eur. J. Biochem. 216, 155-160
3. Errasfa, M. (1991) Characterization of several phospholipase 24. El-Shafei, H.A. and Rezkallah, L.A. (1997) Production, purifica-
activities and diacylglycerol/2-monoacylglycerol lipases in rat tion and characterization of Bacillus lipase. Microbiol. Res. 152,
alveolar macrophages. Biochim. Biophys. Ada 1085, 201-208 199-208
4. Benkirane, M., Meignen, J.M., Boyer, J., and Verine, A. (1989) 25. Iizumi, T., Nakamura, K., Shimada, Y, Sugihara, A., Tominaga,
Lipoprotein lipase in rat heart—I. Characterization of tri-, di-, Y, and Fukase, T. (1991) Cloning, nucleotide sequencing, and
and monoacylglycerol lipase activities in post-heparin effluents. expression in Escherichia coli of a lipase and its activator genes
Comp. Biochem. Physiol. [B] 94, 13-18 from Pseudomonas sp. KWI-56. Agric. Biol. Chem. 55, 2349-
5. Severson, D.L. and Hee-Cheong, M. (1988) Monoacylglycerol 2357
lipase activity in cardiac myocytes. Biochem. Cell Biol. 66, 26. Rua, M.L., Diaz-Maurino, T, Fernandez, V.M., Otero, C, and
1013-1018 Ballesteros, A. (1993) Purification and characterization of two
6. Severson, D.L. and Hee-Cheong, M. (1986) Diacylglycerol lipase distinct Upases from Candida cylindracea. Biochim. Biophys.
and kinase activities in rabbit aorta and coronary microvessels. Ada 1156,181-189
Biochem. Cell Biol. 64, 976-983 27. Imamura, S. and Misaki, H. (1984) A sensitive method for
7. Stam, H., Broekhoven-Schokker, S., and Hiilsmann, W.C. (1986) assay of lipase activity by coupling with P-oxidation enzymes of

J. Biochem.
Monoacylglycerol Lipase from Moderately Thermophilic Bacillus sp. 425

fatty acid in Selected Topics in Clinical Enzymology (Werner, M. Biophys. Ada 1214, 43-53
and Goldberg, D.M., eds.) Vol. 2, pp. 73-77, Walter de Gruyter, 29. Dartois, V, Baulard, A., Schanck, K., and Colson, C. (1992)
Berlin New York Cloning, nucleotide sequence and expression in Escherichia coli
28. Schmidt-Dannert, C, Sztajer, H., Stocklein, W, Menge, U., and of a lipase gene from Bacillus subtilis 168. Biochim. Biophys.
Schmid, R.D. (1994) Screening, purification and properties of a Ada 1131, 253-260
thermophilic lipase from Bacillus thermocatenulatus. Biochim.

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