Partial purification and characterization of acylester hydrolase from Lupinus mutabilis

Article abstract of DOI:10.1007/s11746-999-0089-0

An alkaline acylester hydrolase was partially purified from germinated seeds of Lupinus mutabilis. Hydrolytic activity was absent in the crude extract of ungerminated lupine seed, but it increased and peaked at the fourth day in the germinating seedling.

Full text of DOI:10.1007/s11746-999-0089-0

J9072
Partial Purification and Characterization of Acylester
Hydrolase from Lupinus mutabilis
Walter Anduaga, Ingemar Svensson, Patrick Adlercreutz*, and Bo Mattiasson
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, S-221 00 Lund, Sweden
ABSTRACT: An alkaline acylester hydrolase was partially pu- have been microorganisms such as mold and yeast. Most li-
rified from germinated seeds of Lupinus mutabilis. Hydrolytic pases are reportedly unstable at alkaline conditions in the
activity was absent in the crude extract of ungerminated lupine
seed, but it increased and peaked at the fourth day in the ger-
minating seedling. The purification scheme involved homoge-
nization, centrifugation, acetone precipitation, anion exchange
chromatography, pH precipitation, and hydrophobic interac-
tion chromatography. The acylester hydrolase was purified 126-
fold, and the overall activity yield was 10%. The molecular
weight estimated by sodium dodecylsulfate-polyacrylamide gel
electrophoresis was 60 kDa. The enzyme had an isoelectric
presence of anionic surfactants (6). Lipase from plants has
been studied only to a minor extent (7). Germinating oilseeds
are being explored as a possible source of lipase for the
biotechnological processing of oils and fats (8). Seeds gener-
ally contain starch, proteins, and triacylglycerols as their food
reserve for germination. Most of the investigations on lipase
have been carried out on oleaginous seeds. In oil seeds, lipase
activity is generally expressed during germination (8–10).
point of 6.2 and showed maximal activity at pH 8.0. The en- The utilization of the storage fats is initiated by hydrolysis of
zyme showed good stability between pH 5.0 and 9.0. In the pH triacylglycerols to free fatty acids and glycerol by the lipase.
range 7.0–7.5, enzyme precipitation was observed. The enzyme These intermediate products are then converted to sucrose by
was stable from 0 to 25°C for 5 h and at 45°C lost 50% of its ac-
tivity in the same period of time. At higher temperatures, the en-
zyme showed low thermal stability. However, the highest ini-
tial activity was found to be at 45°C. Nonionic surfactants and
cholic acid enhanced the activity of the enzyme. The activity
was reduced by the addition of toluene and isooctane and in-
creased by the addition of diethyl ether, acetonitrile, methanol,
and pyridine. The activity was reduced by 37% in the presence
of 1 mM Cu2 ions. The enzyme-hydrolyzed triolein showing
no positional specificity.
a long gluconeogenic pathway (11,12) for the support of plant
growth. Lupinus mutabilis is a major crop in the Andean re-
gions of Perú, Bolivia, and the northern Chile. The nutritional
properties and high fat content (15–30%) of lupines are well
documented (13–17). This study describes the extraction, pu-
rification, and properties of an acylester hydrolase from ger-
minated seeds of lupine.
+
MATERIALS AND METHODS
Paper no. J9072 in JAOCS 76, 1157–1162 (October 1999).
Lupinus mutabilis seeds were purchased at a Sunday market in
Cuzco, Perú. Phenyl Sepharose and diethylaminoethyl (DEAE)
Sephadex were obtained from Pharmacia-LKB (Sollentuna,
Sweden), thin-layer chromatography (TLC) plates and Silica
gel 60 were obtained from Merck (Darmstadt, Germany).
KEY WORDS: Acylester hydrolase, Lupinus mutabilis, purifi-
cation.
Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) is an en- p-Nitrophenyl (pNP) esters, the dye Fast garnet GBC base, and
zyme widely present in animals, plants, and microorganisms. α-naphthyl acetate were from Sigma (St. Louis, MO). All
This type of enzyme has become the subject of intense re- reagents and chemicals used were of analytical grade.
search during the last decade, especially due to its potential
Germination of lupine seeds. The seeds were washed, and
applications in industry (1), in synthetic reactions (2), and in soaked for 20 min in distilled water, then wrapped with wet
washing processes (3). In this context, the search for new li- cotton and paper and placed in an incubator. The germination
pases, with new and better specificities, and the technologies was carried out in darkness at 24°C with a relative humidity
for their use are driving these investigations. Lipases with of 80–100%. Germinated seeds with similar root length were
high activity levels at alkaline conditions and with anionic harvested every day for 7 d. They were then weighed,
surfactants present are very useful as potential detergent in- counted, and stored at −20°C.
gredients (4,5). For these applications, the sources of lipase
Extraction of acylester hydrolase. The extraction was car-
ried out at 4°C and the entire germinating seed of lupine was
used. Seedlings (50 g) were washed three times with distilled
water and homogenized in 200 mL of grinding medium using
a household mixer (Braun, Germany). The grinding medium
*
To whom correspondence should be addressed at Department of Biotech-
nology, Center for Chemistry and Chemical Engineering, Lund University,
P.O. Box 124, S-221 00 Lund, Sweden.
E-mail: Patrick.Adlercreutz@biotek.lu.se
Copyright © 1999 by AOCS Press
1157
JAOCS, Vol. 76, no. 10 (1999)

1158
W. ANDUAGA ET AL.
contained: 0.5 M sucrose; 1 mM EDTA; 1 mM MgCl ; 2 mM
Hydrophobic interaction chromatography. A phenyl
2
dithiothreitol; and 150 mM, pH 5.0 citrate buffer or 150 mM, Sepharose column 50 × 2 cm was used for hydrophobic inter-
pH 7.0 or 9.0 Tris-HCl buffer. The homogenate was filtered action chromatography. It was preequilibrated with 10 mM
through a piece of Nitex Cloth (Petko, Elmsford, NY) with a sodium citrate buffer (pH 6.0) with 2.0 M sodium sulfate. The
pore size of 20 × 20 µm. The filtrate was centrifuged at sample (100 mg of protein) loaded onto the column was from
1
3,000 × g for 20 min at 10°C. Three fractions were obtained; the 75% (NH ) SO saturation pellet dissolved in 5 mL of 10
4 2 4
an upper fat layer (spherisomal fraction), a pellet of particu- mM sodium citrate buffer (pH 6.0) with 2.0 M sodium
late material, and a liquid fraction (18). The fat layer was sulfate. The column was washed with the same buffer to elute
carefully removed with a spatula and the liquid supernatant unbound material. The bound protein was then eluted
separated from the pellet. The extracted liquid was used as stepwise with lowering concentrations of sodium sulfate
the crude source of acylester hydrolase.
in the same buffer (1.5, 1.0, 0.5, and 0.0 M). The absorbance
Enzyme activity. Enzyme activity was determined with of the eluate was monitored at 280 nm using a Uvicord de-
p-nitrophenyl acetate (pNPA) as substrate which is hydro- tector from LKB (Sollentana, Sweden). The flow rate
lyzed to p-nitrophenol sodium (pNP). A 10-µL solution of was 1.5 mL/min. Sixty fractions of 5 mL each were collected,
100 mM pNPA in methanol was mixed well with 2 mL phos- and protein content and acylester hydrolase activity were
phate buffer (20 mM, pH 8.0) in a 3-mL cuvette. The reaction assayed.
was started by adding 50 µL of enzyme solution. The forma-
SDS-polyacrylamide gel electrophoresis (PAGE). SDS-
tion of pNP was measured as an increase in absorbance at 410 PAGE of protein fractions from different stages of purification
nm. The reaction rate was linear for the entire time interval of was performed using a 12.5% acrylamide gel with 5% stack-
2–3 min. One unit of acylester hydrolase activity was defined ing gel according to Laemmli (21), in a Bio-Rad Mini Protean
as the amount of enzyme that released 1.0 µmol of pNP per apparatus. Protein was visualized by staining with Coomassie
min. The activity observed was proportional to the amount of Brilliant Blue R (Sigma Chemical Co.).
enzyme added.
Native PAGE. Nondenaturing electrophoresis for the en-
Protein measurement. Quantification of proteins was car- zyme was performed in the Mini Protean unit at 12°C using a
ried out using the bicinchoninic acid method (19) with bovine low-pH discontinuous system (acetic acid-KOH), with a
serum albumin (BSA) as standard.
stacking gel buffer at pH 6.5 (5% acrylamide), resolving gel
Purification of acylester hydrolase. To the liquid fraction buffer at pH 4.5 (12.5% acrylamide), and reservoir buffer of
obtained above (pH 7.0 unless otherwise noted) was added acetic acid-β-alanine (pH 4.5). The system operated with the
6
0 vol% cold acetone (−20°C). The mixture was left to stand cathode at the bottom of the gel. The electrophoresis run
for 1 h to allow protein precipitation to occur. The precipitate lasted almost 2 h. Protein was stained as for SDS-PAGE.
was separated by centrifugation at 8,000 × g for 10 min. The Activity visualization of lipase on native PAGE-gel. After
pellet was then collected, desiccated overnight, and ground. electrophoresis on acidic native gels, acylester hydrolase ac-
The precipitated protein (10 g) was dissolved in 50 mL of 10 tivity was observed as follows: A substrate coupler-staining
mM sodium citrate buffer (pH 6.0). DEAE-Sephadex (2 g) solution was prepared by dissolving 20 mg of α-naphthylace-
was then added, followed by incubation at 4°C, for 20 min on tate (α-NA) in 4 mL of N,N-dimethyl formamide (DMF) and
a reciprocal mixer. The mixture was filtered, and protein con- then added to a 36-mL solution of 20 mg Fast garnet GBC
tent and the acylester hydrolase activity of the filtrate were base in 0.05 M acetic acid-KOH buffer (pH 5.5). The gel was
determined prior to lyophilization.
incubated in 0.1 M acetic acid-KOH buffer (pH 5.5) for 30
pH precipitation. Lyophilized enzyme (78 mg) from the min at 37°C. It was then placed in a freshly prepared substrate
previous DEAE-Sephadex treatment was dissolved in 2 mL coupler-staining solution for 1 h at 37°C. The reaction was
Tris-HCl buffer 10 mM, pH 7.2, and allowed to stand at 4°C stopped by decanting the coupler solution and covering the
for 1 h before centrifugation (5,000 × g for 5 min). The pre- gel with 2% acetic acid.
cipitate containing the acylester hydrolase activity was then
Isoelectric focusing. The isoelectric point (pI) of purified
dissolved in 10 mM citrate buffer (pH 4.0) followed by incu- lupine enzyme was determined using a free solution isoelec-
bation at 4°C for 1 h before centrifugation (5,000 × g for 5 tric focusing apparatus (Rotofor cell, Bio-Rad). In a first run,
min). The acylester hydrolase activity and protein contents Biolyte 3/10 was used as the carrier ampholyte, giving an op-
were determined on the precipitates and the supernatants.
erating range of 3.8–9.3. For the second run, a carrier am-
Ammonium sulfate precipitation. Protein present in the su- pholyte with a operating range of 5.0–8.0 was used. Both am-
pernatant in 10 mM sodium citrate buffer (pH 4.0) was pre- pholytes were from Bio-Rad. The isoelectric focusing was
cipitated with (NH ) SO at 35% of saturation and 4°C for 1 performed according to the instructions provided by the man-
4
2
4
h, then after centrifugation at 5,000 × g for 5 min, the super- ufacturer. Lupine acylester hydrolase sample (2 mL, 10 mg
natant was removed from the pellet. The procedure was re- protein) was used for the procedure. The enzyme was moni-
peated at 55, 75, and 100% saturation. At the end, four differ- tored by both activity and protein measurements.
ent pellets were obtained which were directly lyophilized and
aliquots were dissolved in 10 mM sodium citrate buffer (pH olein, and oleic acid, samples were immediately extracted
.0); and the activity was assayed. with diethylether, then dried and derivatized by the addition
Gas chromatography. For the analysis of monoolein, di-
6
JAOCS, Vol. 76, no. 10 (1999)

ACYLESTER HYDROLASE FROM LUPINUS MUTABILIS
1159
of 10 µL of N-methyl-N-trimethylsilyl-heptafluoro-butyr- × 0.2 mm) plates were first washed by eluting with ace-
amine (MSHFBA). The derivatization reagent converts all tone/water (60:40 vol/vol) as the mobile phase and dried.
hydroxyl groups to the corresponding trimethylsilyl ether. They were then dipped in boric acid, 50 g/L in ethanol, and
After the addition of MSHFBA, the samples were incubated dried at 105°C for 1 h and then kept dessicated until used for
for 15 min at room temperature and were then diluted with 1 analyzing samples from fat hydrolysis.
mL hexane. Dried ethanol (15 µL) was added to react with
any excess of MSHFBA. Analysis was carried out on a Var-
ian 3400 gas chromatograph equipped with an 8035 auto sam-
RESULTS AND DISCUSSION
pler and a flame-ionization detector. An 8-meter DB-1 col- The highest acylester hydrolase activity was expressed after
umn (0.32 mm i.d., 0.15 µm film thickness) from J&W (Fol- 4 to 5 d of germination. When grinding the seedlings, the
som, CA) was fitted to the high-performance insert of the grinding medium with Tris/HCl buffer (pH 7.0) showed the
injector. Helium was used as a carrier gas at constant pressure best performance for extracting and/or expressing the
(
55 KPa for analysis of glyceride species). The column was acylester hydrolase activity (Fig. 1). The lupine-acylester hy-
held at 100°C for 3 min, then the temperature was increased drolase is present in germinated seeds, but not in ungermi-
to 220°C at 13°C /min and held at 220°C for 3.5 min. The de- nated ones. This finding agrees with the characteristic pattern
tector temperature was 350°C, 2-monoolein eluted before 1- of oilseed lipase (10,22)
monoolein and baseline separation was obtained. Standards
for 2-mono, 1,3- and 1,2-diolein were used, and tricaproin of lupine acylester hydrolase is summarized in Table 1. The
was used as internal standard. precipitation with acetone increased the specific activity four-
Purification of lupine enzyme. The purification sequence
Fractionation of the lupine fats. Dried and ground spheriso- fold. During the DEAE-Sephadex treatment, the enzyme re-
mal fraction (20 g) from the homogenized seedlings was dis- mained in the supernatant and the activity yield increased
solved in hexane, and the free fatty acids were washed away 100%, suggesting that some inhibitors were adsorbed onto the
by extraction with methanol/bicarbonate buffer (1:1, vol/vol, resin. Precipitation at pH 7.2 provided an important increase
pH 10.0). Hexane was evaporated and the oil was loaded onto of the specific activity. The enzyme was present in the pre-
a silica gel 60 column (35 × 4 cm) previously equilibrated with cipitate. After precipitation at pH 4.0, the activity was found
hexane, and the triacylglycerols were eluted with hexane/di- in the supernatant, and the specific activity was doubled by
ethylether (70:30 vol/vol). The fractions collected were the precipitation of other proteins. Ammonium sulfate pre-
checked by TLC, fractions which contained lupine-triacylglyc- cipitation was evaluated. For the purification scheme, precip-
erols were pooled and the solvent evaporated.
itation of the enzyme between 55 and 75% saturation was
Substrate specificity. The hydrolysis of triacylglycerol was chosen. Phenyl Sepharose chromatography worked success-
carried out by mixing 250 µL of solution A: 100 mM fully only when 2 M sodium sulfate favored the hydrophobic
monoacid triacylglycerol with one of the fatty acids; C2:0
,
C3:0, C4:0, C10:0, C12:0, C18:0, C18:1 or C20:0, 10% gum arabic
in water (the mixtures were emulsified with a homogenizer
(
Disp 25, InterMed, Roskilde, Denmark) for 2 min, with 250
µL of solution B: 1% NaCl and 1% deoxycholic acid dis-
solved in 50 mM sodium phosphate buffer (pH 8.0). The re-
action was started by adding 50 µL of enzyme solution. The
mixture was incubated at 40°C, at 175 rpm on a reciprocal
shaker. Samples were withdrawn, and the hydrolytic products
were extracted with diethylether for TLC and GC analysis.
In the hydrolysis of pNP esters, the reaction mixture was
composed of 0.95 mL of substrate [2.63 mM pNP esters in 50
mM Tris-HCl buffer (pH 8.0) containing 4% Triton X-100].
The reaction was started by adding 50 µL of acylester hydro-
lase solution. It was then incubated with stirring at 37°C for
1
5 min. The reaction was stopped by the addition of 2.0 mL
of acetone and the absorbance was measured at 410 nm.
TLC. Silica gel 60 F 254 plates were used for monitoring
the progress of the lupine-fat fractionation and the hydrolysis
of the triacylglyceride. Mobile phases were: hexane/ether
(
70:30, vol/vol) and chloroform/acetone/acetic acid (88:12:1,
vol/vol/vol), respectively. The detection was done by dipping
in sulfuric acid/ethanol (15:85, vol/vol), and spots were visu-
alized by heating. For studies of the positional specificity of
FIG. 1. Acylester hydrolase activity during germination. Extraction with
0-mM grinding medium buffers. Citrate buffer (pH 5.0) (■); Tris/HCl
5
lupine acylester hydrolase on TLC, silica gel 60 (10 × 20 cm buffer at pH 7.0 (●); and 9.0 (▲).
JAOCS, Vol. 76, no. 10 (1999)

1160
W. ANDUAGA ET AL.
TABLE 1
Purification Procedure of Acylester Hydrolase from Lupinus mutabilis Germinating Seed
a
Step
of
purification
Total
activity
(U)
Specific
activityb
(U/mg protein)
Activity
yield
(%)
Protein
(g)
Purification
factor
Crude extract
Acetone precipitation
DEAE-Sephadex
pH 7.2 precipitation
pH 4.0 precipitation
8090
6470
12950
2020
0.9
3.59
6.64
25.9
53.1
100
80
160
25
9
1.8
1.95
0.078
0.034
1.0
4.0
7.4
29
1800
22
59
(
NH ) SO (75%)
4 2 4
precipitation
Phenyl Sepharose
1270
750
83.5
113.3
16
9
0.015
0.0066
93
126
a
Batch of 50 g of germinating seed.
Activity was determined by photometric assay with pNPA as substrate.
b
interaction of acylester hydrolase with phenyl Sepharose. The than 6.0, the stability decreased: around 70% of the activity
enzyme could be eluted with 0.5 M sodium sulfate. The en- remained at pH 7.0 and 8.0, and 40% remained at pH 9.0.
tire purification procedure resulted in 126-fold increase in
specific activity.
Temperature dependence of the initial activity and stabil-
ity. The effect of temperature on the initial activity was estab-
Visualization of the acylester hydrolase activity. The stain- lished by measuring the enzymatic activity with pNPA as sub-
ing of enzyme activity in native acidic PAGE showed one strate in 20 mM Tris-HCl buffer, pH 8.0, at different temper-
band of activity, evidencing the hydrolysis of α-NA by the al- atures (Fig. 3). The enzyme activity was highest around 45°C.
kaline lupine acylester hydrolase. In a previous native elec-
To study the thermal stability of the lupine acylester hy-
trophoresis with basic gel, the band of activity was not ob- drolase, pure and unpurified lupine enzyme was kept for 1 yr
served, obviously due to the fact that the enzyme could not at −20°C. Purified lupine enzyme lost 5% of its activity, but
enter that gel.
the unpurified enzyme did not show any loss of activity. This
Characteristics. The apparent molecular weight of the pu- is similar to the long-term stability found in other plant li-
rified enzyme was estimated to be 60 kD by SDS-PAGE. The pases (8,25). The activity was measured after incubation at
gel showed one main band and four to five very faint bands. different temperatures between 25 and 65°C in 50 mM Tris-
To confirm that the main band (60 kD) was the enzyme, a na-
tive gel electrophoresis was done and the band that gave en-
zyme activity was excised. The enzyme was extracted and an-
alyzed with SDS-PAGE. The latter confirmed that the protein
showing activity in the native gel migrated identically with the
6
0 kD protein in the SDS-PAGE. This molecular weight was
within the range (60–65 kD) of other reported plants lipases
7,23,24). The pI value of this enzyme was estimated to be 6.2
(
using a Rotofor isoelectric focusing apparatus.
Effect of pH on activity and stability. The acylester hydro-
lase activity was measured in different 20-mM buffers of
varying pH (from 5.0 to 8.0) using pNPA as substrate. The
enzyme exhibited the highest activity at pH 8.0. At pH 9.0,
the activity decreased by almost 80%. The other plant lipases,
from rapeseed and mustard, also showed the same pH profile
(
8–10). At low pH, the nitrophenol released is not fully ion-
ized and the molar absorbance drops. This produces errors in
the activity measurements at low pH. At any rate, the enzyme
showed activity at pH 5.0 and 6.0.
The effect of pH on stability was studied by dissolving the
lupine acylester hydrolase in different 50-mM buffers (from
pH 2.0 to 9.0) and incubating for 12 h at 30°C. Initial activity
at time 0 was 100%. The activity analysis was carried out
using pNPA as a substrate in 20 mM sodium phosphate
buffer, at pH 8.0. Figure 2 shows that the enzyme was most
stable at pH 6.0 (maximal residual activity). At pH 2.0 and
FIG. 2. Effect of pH on acylester hydrolase stability. Enzyme was incu-
bated at 30°C for 12 h in various pH buffers (50 mM): glycine/HCl
buffer for pH 2.0 and 3.0 (■), citrate buffer for pH 4.0, 5.0, and 6.0 (●),
3
.0 the enzyme precipitated and lost activity. At a higher pH and Tris/HCl buffer for pH 7.0, 8.0, and 9.0 (▲).
JAOCS, Vol. 76, no. 10 (1999)

ACYLESTER HYDROLASE FROM LUPINUS MUTABILIS
1161
TABLE 2
Substrate Specificity of Lupine Acylester Hydrolase
a
Toward Several p-Nitrophenyl Esters
Relative activity (%)
Substrate
25°C
40°C
p-Nitrophenyl acetate
p-Nitrophenyl propionate
p-Nitrophenyl caprylate
p-Nitrophenyl laurate
p-Nitrophenyl stearate
75
65
63
5
100
85
75
10
5
0
aThe enzyme activity was measured by the spectrophotometric method de-
scribed in the Materials and Methods section.
for this substrate. The results were also confirmed by GC
analysis of the silylated reaction products. Spontaneous isom-
erization of the reaction products was considered negligible
owing to the short reaction times (30 min).
Effect of solvents on acylester hydrolase stability. The ef-
fect of organic solvents on stability was studied with lupine
acylester hydrolase. The remaining activity was measured
FIG. 3. The effect of temperature on the initial activity was established after incubation with various solvents. The enzyme was incu-
by measuring the enzymatic activity with pNPA as substrate in 20 mM bated in 50 mM sodium phosphate buffer (pH 8.0), contain-
Tris/HCl buffer (pH 8.0) at different temperatures.
ing 20% vol/vol of various solvents for 60 min at 30°C. The
residual activities, on samples taken from water phase, were
HCl buffer (pH 8.0). Samples were withdrawn periodically assayed by the spectrophotometric method described in the
and the residual activity was assayed using pNPA as substrate Materials and Methods section. The lupine acylester hydro-
at 25°C. The enzyme was stable at 25°C for 5 h, while at lase was stable over the incubation period with most of the
3
5°C, the activity was reduced by 20% after 5 h. The half- water-miscible solvents added. This behavior was also ob-
lives of the acylester hydrolase at 45 and 55°C were around 5 served for a fungal lipase by Lin et al. (26). The activity in-
and 1 h, respectively. At 65 or 75°C, the half-lives were less creased 50% with diethyl ether, 30% with acetonitrile, 18%
than 0.5 h. A similar temperature stability has been reported with methanol, and 13% with pyridine, whereas toluene and
for a peanut alkaline lipase (7).
Substrate specificity. The enzyme was active on lupine oil, Other solvents that caused less than 10% increase of activity
olive oil, and on almost all triacylglycerols tested: tri-C2:0
were: 3-methyl-3-pentanol, ethyl acetate, 2-propanol, and
isooctane reduced the activity by 50 and 40%, respectively.
,
C3:0, C4:0, C10:0, C12:0, and C18:1. Triolein was the substrate acetone. Solvents that caused less than 20% decrease were:
on which the enzyme showed the best hydrolytic activity, dioxane, tetrahydrofuran, hexane, dichloromethane, di-
evaluated by TLC (data not shown). No hydrolysis was ob- methylsulfoxide, and ethanol.
served with tri-C18:0 or with tri-C20:0
.
Effect of metal ions on acylester hydrolase activity. The ef-
Hydrolysis of pNP esters. The specificity of the lupine fect of various metal ions on activity was studied. The enzyme
acylester hydrolase toward pNPA, p-nitrophenyl propionate, was incubated in 50 mM sodium phosphate buffer (pH 8.0),
p-nitrophenyl caprylate, p-nitrophenyl laureate, and p-nitro- containing various metal chlorides at 1 mM concentration, for
phenyl stearate were investigated (Table 2). The acetate ester 30 min at 30°C. The residual activities were assayed by the
was most rapidly hydrolyzed by lupine acylester hydrolase. spectrophotometric method described in the Materials and
Longer fatty acid chains were also hydrolyzed, but at a lower Methods section. The lupine enzyme was significantly inhib-
2
+
+
2+
rate. Stearic acid ester was not hydrolyzed at 25°C, but at ited (40%) by 1 mM of Cu and less inhibited by Cs , Fe ,
2
+
2+
2+
2+
40°C, a slow hydrolysis was observed.
Sn , Co , Mg , and Ca . Metal ions generally form com-
Positional specificity. The positional specificity of lupine plexes with ionized fatty acids, changing their solubility and
acylester hydrolase on triolein was examined by TLC and gas behavior at the interface. However, the inhibition might in-
chromatography (GC) of the hydrolytic products (26). The volve the catalytic site directly, although alteration of the prop-
TLC analysis on boric acid-impregnated plates showed that erties of the interface must also be considered. Almost no in-
2+
2+
2+
2+
substantial amounts of 1,2-dioleyl glycerol and 2-monooleyl hibition was shown with Cd , Mn , Ni , and Zn ions.
glycerol were formed as reaction products; also 1,3-dioleyl
Effect of detergents. The effect of detergents was tested on
glycerol was formed, but at a lower rate. This suggested that lupine acylester hydrolase activity. The enzyme was incu-
the lupine enzyme could cleave in position 1 (3) faster than bated in 50 mM sodium phosphate buffer (pH 8.0), contain-
in position 2. The enzyme seems to hydrolyze all three posi- ing 0.1% (wt/vol) detergents for 60 min at 30°C. Inhibition
tions in triacylglycerides and can be regarded as unspecific (20%) was observed with the addition of dioctyl sulfosucci-
JAOCS, Vol. 76, no. 10 (1999)

1162
W. ANDUAGA ET AL.
nate, sodium salt. With sodium dodecyl sulfate and sodium 13. Egana, J.I., R. Uauy, X. Cassorla, G. Baweva, and E. Yanez,
Sweet Lupin Protein Quality in Young Men, J. Nutr.
deoxycholate, no effect was observed. All the nonionic sur-
1
22:2341–2347 (1992).
factants tested produced an activating effect: sorbitan tris-
tearate (27%), Tween 80 (19%), and Triton X-100 (10%).
Furthermore, cholic acid, which is known to stimulate pan-
1
4. King, J., C. Aguirre, and S. De Pablo, Funtional Properties of
Lupin Protein Isolates (Lupinus albus cv Multolupa), J. Food
Sci. 50:82–87 (1985).
creatic lipase, stimulated lupine acylester hydrolase activity 15. Dagmia, S.G., D.S. Pettersson, R.R. Bell, and F.V. Flanagan,
Germination Alters the Chemical Composition and Protein
Quality of Lupin Seeds, J. Sci. Food Agric. 60:419–423 (1992).
6. Huesa, J., and J. Lopez, El Aceite de Altramuz (lupino). Su
Aprovechamiento Como Comestible, Aliment. Equipos Tecnol.
3:161–168 (1984).
by 30%. Whether the stimulation of acylester hydrolase ac-
tivity was due to a direct interaction of surfactants with the
enzyme or to an alteration of emulsion properties which in
turn affected activity is not known. Similar results have been
1
reported by others for lipases from different sources (26–28). 17. Gross, R., and E. Baer, in Proyecto Lupino. Informe N˚ 2, Insti-
tutos Nacionales de Salud, Instituto de Nutricion, Lima, Perú,
1
974, p. 86.
ACKNOWLEDGMENTS
1
1
8. Espin, F., and J. Davila, Investigación Tecnológica para Elimi-
nación Industrial de los Alcaloides del Chocho, Politecnica 11,
4
9. Huang, A.H.C., and R.A. Moreau, Lipase in the Storage Tissue
We gratefully acknowledge financial support from Swedish Agency
for research cooperation with Developing Countries SAREC-SIDA
:23–34 (1986).
(Sweden).
of Peanut and Other Oil Seeds During Germination, Planta
1
41:111–116 (1978).
REFERENCES
20. Smith, P.K., R.I. Krohn, and E.K. Hermanson, Measurement of
Protein Using Bicinchoninic Acid, Anal. Biochem. 150:76–85
1
2
3
. Cerletti, P., and M. Duronti, Development of the Lupine Pro-
teins, J. Am. Oil Chem. Soc. 56:460–466 (1979).
(1985).
2
2
1. Laemmli, U.K., Cleavage of Structural Proteins During the As-
. Mukherjee, K.D., Lipase-Catalyzed Reactions for Modification
of Fats and Other Lipids, Biocatalysis 3:277–293 (1990).
. Fuiji, T., T. Tatara, and N. Minagawa, Studies on Applications
of Lipolytic Enzyme in Detergency. I. Effect of Lipase from
Candida cylindracea on Removal of Olive Oil from Cotton Fab-
ric, J. Jpn. Oil Chem. Soc. 63:796–799 (1986).
sembly of the Head of Bacteriophage T4, Nature 227:680–685
(1970).
2. Lin, Y.-H., R. Moreau, and A.H.C. Huang, Involvement of Gly-
oxysomal Lipase in the Hydrolysis of Storage Triacylglycerols
in the Cotyledons of Soybean Seedlings, Plant Physiol.
7
0:108–112 (1982).
4
. Hashimoto, T., T. Fuiji, and T. Kawase, Studies on Applications
of Lipolytic Enzymes in Detergency. III. Removal of Triolein
and Its Hydrolysates from Cotton Fabric, Ibid. 34:606–612
2
3. Hills, M.J., and H. Beevers, An Antibody to the Castor Bean
Glyoxysomal Lipase (62 kD) Also Binds to a 62 kD Protein in
Extracts from Many Young Oilseed Plants, Ibid. 85:1084–1088
(
1985).
(1987).
5
. Kotani, T., T. Fuiji, and H. Okuyama, Studies on Removal of a
2
2
2
2
2
4. Downey, W.K., and P. Andrews, Gel Filtration Applied to the
Fatty Soil Mixture of Triglyceride and Fatty Acid, Ibid.
Study of Lipases and Other Esterases, Biochem. J. 94:642–650
2
8:914–918 (1979).
(1965).
6
7
8
. Andree, H., W.R. Muller, and R.D. Schmid, Lipases as Deter-
gents Components, J. Appl. Biochem. 2:218–229 (1980).
. Sanders, T., and H. Pattee, Peanut Alkaline Lipase, Lipids 10:
5. Ory, R.L., A.J. St. Angelo, and A.M. Altschul, The Acid Lipase
of the Castor Bean: Properties and Substrate Specificity, Ibid.
3
:99–105 (1962).
5
0–54 (1974).
6. Lin, S.-F., J.-C. Lee, and C.-M. Chiou, Purification and Charac-
terization of a Lipase from Neurospora sp. TT-241, J. Am. Oil
Chem. Soc. 73:739–745 (1996).
7. Yamamoto, K., and N. Furiwara, Purification and Some Proper-
ties of a Castor-Oil-Hydrolyzing Lipase from Pseudomonas sp.,
Agric. Biol. Chem. 52:3015–3021 (1988).
8. Kundu, M., M. Basu, M. Guhhait, and P. Chakrabarti, Isolation
and Characterization of an Extracellular Lipase from the Coni-
dia of Neurospora crassa, J. Gen. Microbiol. 133:149–153
. Hassanien, F.R., and K.D. Mukherjee, Isolation of Lipase from
Germinating Oilseeds for Biotechnological Processes, J. Am.
Oil Chem. Soc. 63:893–897 (1986).
9
. Theimer, R.R., and I. Rosnitschek, Development and Intracellu-
lar Localization of Lipase Activity in Rapeseed (Brassica napus
L.) Cotyledons, Planta 139:249–256 (1978).
1
0. Lin, Y.-H., and A.H.C. Huang, Lipase in Lipid Bodies of
Cotyledons of Rape and Mustard Seedlings, Arch. Biochem.
Biophys. 225:360–369 (1983).
(1987).
1
1. Huang, S.H.C., Enzymes of Glycerol Metabolism in the Storage
Tissues of Fatty Seedlings, Plant Physiol. 55:555–558 (1975).
2. Hutton, D., and P.K. Stumpf, Characterization of the β-Oxida-
tion Systems from Maturing and Germinating Castor Bean
Seeds, Plant Physiol. 44:508–519 (1969).
1
[Received November 3, 1998; accepted May 17, 1999]
JAOCS, Vol. 76, no. 10 (1999)