RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+.. Previously, we reported on th
Trang 1Purification and properties of the extracellular lipase, LipA,
Erick A Snellman1,2, Elise R Sullivan1,3and Rita R Colwell1,4
1
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, MD, USA;2HQ USAFA/DFB, USAF Academy,
CO, USA;3Department of Microbiology, University of New Hampshire, USA;4Department of Cell and Molecular Biology, University of Maryland, USA
An extracellular lipase, LipA, extracted from Acinetobacter
sp RAG-1 grown on hexadecane was purified and
proper-ties of the enzyme investigated The enzyme is released into
the growth medium during the transition to stationary
phase The lipase was harvested from cells grown to
sta-tionary phase, and purified with 22% yield and > 10-fold
purification The protein demonstrates little affinity for
anion exchange resins, with contaminating proteins removed
by passing crude supernatants over a Mono Q column The
lipase was bound to a butyl Sepharose column and eluted in
a Triton X-100 gradient The molecular mass (33 kDa) was
determined employing SDS/PAGE LipA was found to be
stable at pH 5.8–9.0, with optimal activity at 9.0 The lipase
remained active at temperatures up to 70C, with maximal
activity observed at 55C LipA is active against a wide
range of fatty acid esters of p-nitrophenyl, but preferentially
attacks medium length acyl chains (C6, C8) The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+ The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine RAG-1 lipase offers potential for use as a biocatalyst Keywords: lipase; LipA; Acinetobacter sp RAG-1; protein purification; zymogram
Lipases are glycerol ester hydrolases (EC 3.1.1.3) that
catalyze the hydrolysis of triacylglycerols to free fatty acids
and glycerol They resemble esterases in catalytic activity,
but differ in that their substrates are water-insoluble fats
containing medium to long fatty acyl chains [1] Lipases are
further distinguished from esterases in that they are
activated at the substrate–water interface [2] Interest in
lipases has increased recently due to their recognition as
important virulence factors [3] and their biotechnological
potential Lipases have proven to be versatile enzymes in
nonaqueous solvent systems in which they catalyze the
synthesis of a variety of acylglycerols and specialized esters
via transesterification Lipases demonstrating high activity
under alkaline conditions are used as additives in detergents,
one of the largest industrial uses of these enzymes [4]
Lipase-catalyzed synthesis of structured triacylglycerols
comprised of both long and medium chain fatty acids has
been investigated as a means of providing single substitutes
for mixed acylglycerides in dietary applications [5] In both
hydrolysis and synthesis reactions, lipases demonstrate stereo- and regio-selectivity, making them good candidates for production of optically active compounds used in the pharmaceutical and agricultural industries
Previously, we reported on the cloning and sequence of an extracellular lipase (LipA) from Acinetobacter sp RAG-1, which contained several conserved regions common to bacterial lipases [6] This strain has also been characterized with respect to its production of a powerful emulsifying agent, termed emulsan [7,8] Emulsan is a heteropolysaccha-ride complex produced as a capsule in cells grown on hydrocarbons and ethanol as sole carbon sources and is released into the growth medium during transition to stationary phase [8,11] The bioemulsifier is composed of a polysaccharide backbone with attached fatty acids and noncovalently bound proteins [9,10] Because LipA is also produced during growth on hydrocarbons [6], despite the fact that alkanes are not lipase substrates, we are currently investigating the potential role of LipA in facilitating emulsification by interaction with emulsan To this end we report here the purification and characterization of LipA and discuss its potential as a biocatalyst in synthesis reactions and
in the context of related biotechnological applications
M A T E R I A L S A N D M E T H O D S
Media and culture conditions Acinetobacter sp RAG-1 (ATCC 31012) recovered from frozen stock ()80 C) was used to inoculate Spirit Blue agar (Difco, Liverpool, Australia) cultures, which were incubated
Correspondence to R R Colwell, Center of Marine Biotechnology,
Suite 236, Columbus Center, 701 East Pratt Street,
Baltimore, Maryland, USA, 21202.
Fax: + 1 703 292 9232, Tel.: + 1 703 292 9232,
E-mail: rcolwell@umbi.umd.edu
Abbreviations: LNPS, low nitrogen, phosphorous, sulfur; pNPP,
p-nitrophenyl palmitate; pNP, p-nitrophenol; HIC, hydrophobic
interaction chromatography.
(Received 5 April 2002, revised 23 July 2002,
accepted 6 September 2002)
Trang 2overnight at 37C Single colonies were selected for
inocu-lation of 100 mL low nitrogen, phosphorous, sulfur (LNPS)
medium consisting of (per litre): KH2PO4, 3.3 g; Na2HPO4,
2.2 g; Na2SO4, 1.0 g; NH4NO3, 1.0 g; NaCl, 5.0 g; MgSO4,
0.29 g; CaCl2, 0.05 g; FeSO4, 1 mg (pH 7.0) Hexadecane
(10 mM) was employed as carbon source Inocula were
grown overnight at 30C, with shaking at 200 r.p.m in a
rotary incubator/shaker (New Brunswick Scientific, Edison,
NJ) Aliquots from overnight cultures were transferred to
fresh LNPS amended with hexadecane and used for growth
studies and lipase production Cultures were grown (as
above) for 48 h prior to harvest Extracellular lipase activity
in these cultures ranged from 0.2 to 0.35 units (U) mL)1
Lipase assay
Lipase activity was measured by hydrolysis of p-nitrophenyl
palmitate (pNPP) in deoxycholate buffer, as described
elsewhere [6,12] All assay reagents were purchased from
Sigma (St Louis, MO) Samples (20 lL)500 lL) were
added to prewarmed (30C) phosphate buffer (50 mM,
pH 8) containing 0.2% (w/v) sodium deoxycholate and
0.1% (w/v) gum arabic, final volume 3.0 mL The mixture
was incubated for 5 min at 30C pNPP (0.30 mM final
concentration) was added and the mixture shaken, allowing
the reaction to proceed for 3 min Lipase activity was
determined by the rate of p-nitrophenol production (pNP),
measured at 405 nm in a model DU640 spectrophotometer
(Beckman Coulter, Fullerton, CA) Lipolytic activity was
determined, using substrate free blanks as control The
reaction rate was calculated from the slope of the
absorb-ance curve, using software installed by the manufacturer
(Beckman Coulter) The extinction coefficient under the
conditions described was 17454 LÆmolÆcm)1[6] One unit of
enzyme activity is defined as the amount of enzyme forming
1 lmol of pNP min)1 Lipase specific activity was expressed
as unitsÆmg protein)1 When examining the effect of
tem-perature on activity, enzyme preparations were incubated at
different temperatures for 5 min and assayed at the
incubation temperature
During growth studies, cell bound and cell free lipase
activities were determined as follows: cells were pelleted,
washed twice, and resuspended in sterile LNPS prior to
assay Supernatants were filtered (0.2 lm Tuffryn
mem-brane, Gelman Laboratories, Ann Arbor, MI, USA) and
assayed separately
Protein concentration
Protein was measured using the method of Bradford [13],
with BSA as standard Detergent-compatible BCA protein
assay (Pierce, Rockford, Il) was used to determine protein
concentration in samples containing Triton X-100 Total
protein of cellular fractions was determined after cell
disruption by sonication (3· 30 s) using a Branson model
450 sonicator fitted with a 1.0-mm microtip Protein
concentration was routinely used as a measure of cell
growth in hydrophobic media [14,15]
Lipase purification
After incubation for 48 h in LNPS amended with 10 mM
hexadecane, cells were removed by centrifugation (10 000 g)
at 4C and the supernatants pooled To increase the yield of lipase, 2 mMCaCl2 and 2 mMMgCl2were added during magnetic stirring and the crude supernatant centrifuged a second time To remove residual hexadecane, the combined sample was allowed to stand for 30 min prior to passage through a coarse glass fiber filter, after which the sample was filtered (0.2 lm)
Supernatants were concentrated by ultrafiltration, using
an Amicon RA2000 filter unit fitted with an S1Y10 spiral membrane (10 000 MW, 20 psi) and the sample volume reduced approximately 10–15-fold to 200 mL After con-centration, a serine protease inhibitor, phenylmethanesulfo-nyl fluoride, was added (0.2 mM) to reduce loss from proteolytic activity Concentrated supernatants were ultra-centrifuged at 141 000 g (1 h) at 4C Supernatants con-taining lipase were divided into 35 mL aliquots and stored
at)80 C prior to chromatography
Preliminary experiments to investigate binding properties
of the lipase, using ion exchange resins, showed little (10%) affinity for the matrices under the conditions employed However, other proteins were effectively bound, yielding significant purification Therefore, anion exchange was employed as a preliminary step to hydrophobic interaction chromatography (HIC) Samples were dialyzed overnight against 20 mM Tris/HCl buffer (pH 8.0), followed by passage through an Econo-Pac Mono Q cartridge (5 mL) (Bio-Rad, Hercules, CA) at approximately 1 mL min)1, prior to loading on the hydrophobic matrix
HIC methods were employed as described elsewhere [16,17] An equal volume of Buffer 1 (30 mMTris/HCl; 2 mM CaCl2; 2 mMMgCl2; 0.5MNaCl) was added to supernatant that had equilibrated at room temperature for 30 min Aliquots were added to 25 mL of Butyl Sepharose Fast Flow
4 hydrophobic medium (Amersham Pharmacia Biotech, Piscataway, NJ, USA) equilibrated in the same buffer Equilibration of HIC gel and samples at room temperature allowed for more than 90% of the lipase to be bound The protein slurry was degassed and loaded onto a glass Econo-Column (Bio-Rad) fitted with a column adaptor, for a total column volume of 20–25 mL Lipase was eluted using two linear gradient profiles and flow rate of 1 mLÆmin)1 Two column volumes of Buffer 1 were passed through the column, followed by two volumes of a decreasing salt gradient (0.5M)0MNaCl in Buffer 1) and washed in six volumes of the same buffer without salt A second gradient of 10 column volumes of Triton X-100 (0–1.0%) in 30 mM Tris/HCl
pH 8.0, followed by an additional 60 mL 1% Triton X-100, was used to elute LipA and other highly hydrophobic proteins Absorbance (280 nm) was monitored and fractions (8.0 mL) collected from the detergent gradient were assayed for lipase activity Fractions containing pure lipase, based on SDS/PAGE results, were pooled and concentrated, using ultrafiltration (PM10 membrane, Millipore, Bedford, MA)
or an Ultrafree-4 centrifugal filter unit (10 000 MW cut-off, Millipore) and stored at)80 C
Detergent removal Excess Triton X-100 was removed from the protein preparations by incubating samples at 4C with polymeric absorbent Amberlite XAD-2 (Sigma, St Louis, MO) The absorbent was cleaned following the manufacturer’s instructions and fine particles removed by siphoning after
Trang 3each of several washes in distilled water The absorbent was
equilibrated in 30 mM Tris/HCl at pH 8.0 prior to use
Under these conditions, approximately 50 mg detergent was
removed per gram (wet) of absorbent The amount of
detergent (%) remaining was calculated by plotting A289of
samples against a standard curve The absorbent was
removed from the supernatant by centrifugation (8000 g,
30 min) at 4C
Electrophoresis and preparation of zymograms
SDS/PAGE gel electrophoresis was performed, according
to the methods of Laemmli [18] Protein samples were
prepared in sample buffer (0.5M Tris/HCl, pH 6.8)
con-taining 2% SDS, 2.5% 2-mercaptoethanol, 0.1%
bromo-phenol blue, and 8Murea Electrophoresis was performed
using 12% polyacrylamide gels containing 5Murea
Broad-range molecular weight standards (Bio-Rad) were used for
mass determinations Proteins were stained with Coomassie
Blue R-250
Nondenaturing or native PAGE was performed using the
discontinuous gel system of Orstein [19] and Davis [20] Gels
were cast with a 4% stacking gel and 6% resolving gel
Proteins were allowed to stack at 80 mV and separate at
160 mV Prior to incubation with activity gels, native gels
were rinsed three times with distilled water and equilibrated
in 30 mMTris/HCl pH 8.0 containing 1% Triton X-100 for
30 min at 25C
IEF determination of LipA pI was performed using
precast IEF gels, according to manufacturer’s instructions
(Bio-Rad) The anode and cathode buffers were 7 mM
phosphoric acid and 20 mMlysine/20 mMarginine,
respect-ively IEF standards were purchased from Sigma and
consisted of trypsin inhibitor (pI 4.6); carbonic anhydrase
(pI 6.6); and lentil lectin (pI 8.2, 8.6, 8.8)
Zymograms were accomplished by two methods LipA
activity against triolein (olive oil) was demonstrated
accord-ing to the method of Gilbert et al [21] In summary, gel
overlays were prepared from a 5% olive oil (Sigma)
emulsion in 50 mM Tris/HCl (pH 8.5) containing 0.01%
(w/v) Victoria Blue B dye (pH indicator) and 1.3% agarose
(Fisher Scientific, Pittsburgh, PA) Victoria Blue B was
added as a solution in 70% ethanol Zymograms were also
prepared from an emulsion of 1% tricaprylin (C8:0) in
25 mMTris/HCl and 5 mMCaCl2(pH 8.0) [22] Gels were
cast between glass plates at 4C Activity staining was
accomplished by overlaying native gels with the zymograms
in a closed glass dish and incubating at 37C from 4 to
16 h The incubation chamber was kept humid by addition
of paper toweling saturated with Tris buffer Lipase activity
against olive oil was recorded by appearance of a dark blue
band Positive indicator of tricaprylin hydrolysis was
recorded by appearance of a zone of clearing
Effect of pH on lipase activity and stability
Concentrated lipase preparations were diluted fourfold in
NaH2PO4-NaOH buffer (50 mM) at various pH values and
incubated at 30C for 1 h Lipase activity was determined
in the same buffer plus 0.1% (w/v) gum arabic To
determine the effect of pH on enzyme stability, concentrated
lipase preparations were diluted fourfold in various buffers
and incubated for 24 h at 20C Buffers (50 m ) used were
sodium acetate (pH 5.0–5.6), Tris/malate (pH 5.8–7.5), Tris/HCl (pH 7.5–8.5), 2-amino-2-methyl-1, 3-propanediol (pH 8.2–9.5), and glycine-NaOH (pH 8.6–10.6)
Substrate specificity LipA activity toward substrates with different acyl chain lengths was determined under standard conditions using various esters of p-nitrophenyl (pNP) Substrates and chain lengths examined were as follows: pNP acetate (C2); pNP butyrate (C4); pNP caproate (C6); pNP caprylate (C8); pNP caprate (C10); pNP laurate (C12); pNP myristate (C14); pNP palmitate (C16); and pNP stearate (C18) Substrate stock solutions (36 lL) were added to the reaction mixture (final concentration, 0.3 mM) containing lipase and the reaction allowed to proceed for 3 min
Effect of Ca2+on lipase stability Results of lipase sequence analysis in our laboratory suggested the presence of a Ca2+-binding site in LipA [6] However, the importance of Ca2+-sequestering to enzyme stability in LipA was not investigated We chose to examine the effect of Ca2+loss on stability by incubating LipA in the presence and absence of Ca2+and examining effects over time Concentrated enzyme preparations in 30 mM Tris/ HCl, pH 8.0 (approximately 3.5 UÆmL)1) were dialyzed overnight against 1 L 50 mM Tris/HCl, pH 8.0 After dialysis, 20 lL aliquots of protein solution were diluted fourfold in 50 mMTris/HCl/2 mMMgCl2, with and without addition of 2 mMCaCl2and incubated at 30C During a 30-h period, samples in triplicate were randomly selected at times indicated and examined for lipase activity
Sensitivity to inhibitors and organic solvents LipA preparations were incubated with various compounds
of potential inhibitory activity Prior to incubation in the presence of these compounds, protein samples were dialyzed overnight against 30 mMTris/HCl pH 7.2 Lipase samples were diluted with stock solutions of inhibitors (final concentrations, 0.1 mM, 1.0 mM, or 10.0 mM) and incuba-ted at 30C for 1 h At the end of the incubation period, residual activity was determined using pNPP as the substrate Stability of LipA in organic solvents was meas-ured in a similar fashion using lyophilized enzyme (0.01 mg) incubated in water miscible solvents (15% and 30%, v/v) in
30 mM Tris/HCl (pH 8.0) for 1 h at 30C Activity remaining (%) was measured under standard conditions
R E S U L T S A N D D I S C U S S I O N
Acinetobacter sp RAG-1 has been shown to produce significant amounts of extracellular lipase when grown in LNPS minimal medium amended with hexadecane [6] However, it was not within the scope of that study to determine the spatial and temporal distribution of the enzyme In this study, in order to determine optimum time
to harvest the lipase for purification, both cell-bound and cell-free lipase activities were measured (Fig 1) The data show extracellular lipase production in RAG-1 is growth phase dependent During exponential growth, little cell-free lipase is detected, but a 10-fold increase in extracellular
Trang 4lipase activity was observed during transition to stationary
phase Similar increases in cell-free lipase activity during
transition to stationary phase have been reported in other
Acinetobacter calcoaceticusstrains grown in more complex
media [12,23] and in minimal medium supplemented with
hexadecane [24] Kok et al [24] reported the growth phase
dependent pattern of extracellular lipase activity in cultures
of A calcoaceticus BD413 and AAC321-1 grown on
hexadecane as the sole carbon and energy source They
found lipase production is primarily regulated by LipA
expression (measured by a-galactosidase expression in the
lipA:lacZstrain) induced only after exponential growth had
ceased, indicating that hexadecane itself did not induce
lipase production Moreover, they suggested that
hexade-cane, or one of its degradation products (hexadecanoic acid)
may repress LipA expression Repression of lipase activity
by fatty acids has also been reported in Pseudomonas
aeruginosa EF2 grown on Tween 80 [25] It would be of
interest to determine if RAG-1 lipase production is also
regulated by fatty acid repression of LipA
Purification
RAG-1 lipase was purified from stationary phase cells
grown in LNPS amended with 10 mM hexadecane as the
sole carbon source Under these conditions, lipase accumu-lates in the medium with no apparent loss of activity, making it suitable for purification LNPS medium supple-mented with various triglycerides as sole carbon sources were also investigated for suitability in lipase purification However, in these media a significant and rapid reduction in activity was noted as exponential growth ceased (data not shown) This phenomenon has been previously reported for
A calcoaceticusBD413 grown in nutrient rich media [16]
In that study, it was suggested that loss of activity was due
to proteolytic degradation that does not occur in a minimal medium amended with hexadecane [16]
LipA was purified 10-fold and 22% yield A summary of the purification data is presented in Table 1 Prior to separation by HIC, supernatant samples were passed through a Mono Q column to remove contaminating proteins LipA was effectively bound to the butyl Sepharose resin at 25C (90% of total lipase activity bound) The lipase was eluted from the HIC matrix in two gradients: decreasing NaCl gradient, followed by increasing detergent (Triton X-100) gradient No appreciable lipase activity was detected in fractions collected under conditions of decreas-ing NaCl concentration LipA is effectively eluted from the hydrophobic matrix only under conditions of increasing detergent concentration Lipase begins to elute from the column at 0.4% Triton X-100 followed by an activity peak
at 0.7% detergent Remaining LipA activity decreased rapidly in 1% Triton X-100, indicating effective elution of the protein under these conditions Fractions containing the highest lipase activity were pooled and examined for purity
by SDS/PAGE Only a single major protein band, whose molecular mass (approximately 33 kDa) is consistent with the molecular mass deduced from the nucleotide sequence
of lipA, was observed in these fractions (Fig 2) The fact that LipA binds butyl Sepharose resins under low salt concentration (0.25M NaCl) and detergent is required to elute the lipase suggests it is hydrophobic in nature We often observed smearing in our gels (65 kDa region), presumably due to lipase association with residual emulsan during purification The lipase may associate with the lipophilic component of emulsan through hydrophic inter-action LipA was determined to have a pI of 5.9 (not shown), in close agreement with the predicted value of 6.2 based on sequence analysis [6]
LipA demonstrates hydrolytic activity toward emulsions
of both medium and long chain triacylglycerols (Fig 3) Areas of olive oil and tricaprylin hydrolysis are clearly seen and correspond with the single protein band stained in native-PAGE gel In these experiments, we found LipA shows a tendency toward aggregation, as some of the lipase molecules failed to enter the gel, with a corresponding positive indication of lipase activity in those areas of the
Fig 1 Growth and lipase production by Acinetobacter sp RAG-1 in
LNPSmedium supplemented with 10 m M hexadecane Growth (r) of
RAG-1, measured as total protein (lgÆmL)1) Cell-free (s) and cell
bound (j) lipase activity (unitsÆmL)1) was determined under standard
conditions One unit of enzyme activity catalyzes the production of
1 lmol of pNPÆmin)1 Values are means of three replicates ± SE.
Table 1 Purification of LipA from Acinetobacter sp RAG-1.
Purification method
Protein (mg)
Activity (units) a
Specific activity (unitsÆmg protein)1)
Purification (fold)
Yield (%)
a
One unit of enzyme activity catalyzes the production of 1 lmole of pNP min)1.bTwo litres of filtered supernatant were concentrated 10-fold to 200 mL.
Trang 5zymograms Other investigators have also reported
aggre-gation of purified lipases to a varying degree [21,26–28]
Application of purified LipA to wells in Spirit Blue agar
indicator plates containing an emulsion of tributyrin also
indicates activity toward this lipidic substrate (not shown)
As LipA is able to hydrolyze long-chain triacylglycerol
esters, it merits classification as a true lipase (E.C 3.1.1.3)
Effect of pH on lipase activity and stability
Figure 4A shows activity of LipA at various pH values,
incubated at 30C with p-NPP as substrate The optimal
pH was found to be approximately 9.0 The enzyme showed
strong activity in a narrow pH range of 8.0–10.0, but
activity decreased rapidly at pH exceeding 10.5 These data
are in agreement with those for alkaline lipases reported for
strains of Pseudomonas [21,28] and Acinetobacter [17,26]
and Bacillus subtilis 168 [27] In comparison, the pH stability
curve of LipA showed that the enzyme is stable under a
wider pH range and slightly more acidic conditions
(Fig 4B) LipA retained 100% activity at pH 5.8–9.0, when
incubated for 24 h at 20C Below pH 5.6, stability of the
molecule decreased sharply The stability data suggest that
the sharp decrease in activity below pH 6 (Fig 4A) was not
because of poor stability (Fig 4B), but may be a result of
titration of the imidazole ring of the active site histidine
Further, as significant activity was retained (80%) after
incubation at pH 5.6, we suggest the dramatic reduction in
stability under more acidic conditions (pH < 5.6) can be
explained by titration of the Ca2+-coordinating Asp
residues, as has been previously suggested [30]
Temperature
The optimal reaction temperature for LipA activity towards
p-nitrophenyl palmitate is 55C (Fig 5) At this
tempera-ture, LipA showed a threefold increase in activity compared
with that at 30C LipA remained active at higher
temperatures, with activity exceeding 1.5-times that
observed at 30C at temperatures up to 70 C The optimal
reaction temperature reported here is higher than that reported for many bacterial lipases under similar experi-mental conditions, although higher temperature optima have been reported for several lipases of Pseudomonas spp [31] Activity at high temperature is a useful characteristic for lipases that are used in detergent formulations and biotransformations
Substrate specificity Substrate specificity of LipA was examined using various fatty acid esters of p-nitrophenyl The enzyme showed activity toward a broad range of acyl chain lengths but maximum activity toward medium length fatty acid esters (C6 and C8) (Fig 6) LipA showed little esterase activity toward the more soluble substrate, p-nitrophenyl acetate (C2) A similar preference for medium chain esters and triacyl glycerols has been reported for lipases purified from Bacillus subtilis 168 and Aeromonas hydrophila [27,32] Lipases and esterases share common substrate specificities However, unlike esterases, lipases often dem-onstrate interfacial activation, i.e a marked increase in activity upon the formation of a lipid–water interface [33]
Fig 3 Zymograms demonstrating LipA activity toward olive oil (A) and tricaprylin (B) emulsions Native PAGE gel was incubated between gels A and B for 16 h at 37 C in a sealed, humid chamber A positive indicator of lipolysis is demonstrated in (A) by the release of oleic acid and its interaction with the pH indicator (Victoria Blue B) and in (B)
by tricaprylin hydrolysis (zone of clearing) Lane 1, mixed marker proteins (negative control); lane 2, ion exchange fraction (0.06 units); lane 3, purified LipA (0.3 units); lane 4, lipase from Chromobacterium viscosum (Sigma) (0.25 units, positive control).
Fig 2 SDS/PAGE of extracellular lipase purified from Acinetobacter
sp RAG-1 Lane 1, molecular mass standards; lane 2, crude
super-natant; lane 3, ion exchange fraction; lane 4, purified LipA (10 lg).
Trang 6Therefore, lipase substrates are typically long chain
(‡ C10) fatty acid esters available in micellar form
[34,35] The properties of LipA reported here are
consis-tent with this description LipA is capable of hydrolyzing long acyl chain triglycerides, as in the case of olive oil emulsion used in zymogram preparations, and demon-strated uniform activity against various water-insoluble esters of p-nitrophenyl (Figs 3 and 6) In addition, results
of LipA sequence analysis demonstrated overall similarity with other bacterial lipases [6] These data confirm LipA classification as a true lipase
Stability of LipA in the presence of Ca2+
The deduced sequence of RAG-1 LipA contains two putative Ca2+-binding residues (Asp240, Asp282) that participate in protein stabilization [6] Comparative sequence analysis showed that residues associated with
Ca2+-binding and protein stabilization are universally conserved in Group I Proteobacterial lipases [6] However, biochemical evidence supporting the presence of a Ca2+ -binding site in LipA was not reported Here, we examine the effect of Ca2+ loss on enzyme activity and discuss its stabilization effect on RAG-1 lipase
LipA was incubated in the presence and absence of 2 mM CaCl2and activity loss was measured over a 30-h period at
30C During this time, enzyme preparations without calcium showed a linear decrease in activity In the absence
of Ca2+, up to 70% of the initial activity is lost, whereas enzyme incubated in the presence of calcium retained 100% activity (Fig 7) The data clearly showed that Ca2+ enhan-ces stability of RAG-1 lipase at 30C We attribute the gradual decrease in activity to slow diffusion of Ca2+from its binding site, resulting in inactivation The mechanism explaining inactivation by Ca2+loss is unclear, but may be attributed to concomitant destabilization in the local struc-ture surrounding the active site histidine [30,36] Enzyme stabilization by calcium has been demonstrated in other studies [16,29,37] Lipase purified from A calcoaceticus AAC323-1 demonstrated a greater loss in activity in the absence of Ca2+, with almost no activity toward p-nitrophenyl palmitate within 3 h [16] Biochemical studies
of lipase prepared from Pseudomonas (Burkholderia) glumae also showed reduction in activity associated with calcium loss [36] Addition of calcium was found to prevent heat inactivation of lipase prepared from P fluorescens MC50 incubated at 60C [37] Collectively, these data comprise
Fig 6 Substrate specificity of LipA Acyl-chain length specificity of purified LipA was determined from its activity toward various esters of p-NP (0.3 m M ) Percentages shown are relative to maximum activity (C 6 ).
Fig 4 The effect of pH on LipA activity (A) and stability (B) (A)
Concentrated lipase samples were diluted fourfold in Na 2 PO 4 -NaOH
buffer (50 m M ), pH adjusted, and incubated for 1 h at 30 C Lipase
activity was assayed in the same buffer using p-NPP as the substrate.
Results expressed as relative percentage of maximal activity (pH 9.0).
(B) Lipase preparations were incubated for 24 h at 20 C in selected
buffers and remaining activity (% of initial activity) determined under
standard conditions (pH 8.0) Buffers: (d), sodium acetate (pH 5.0–
5.6); (h), Tris/malate (pH 5.8–7.5); (m), Tris/HCl (pH 7.5–8.5); (·),
2-amino-2-methyl-1, 3-propanediol (pH 8.2–9.5); (e), glycine-NaOH
(pH 8.6–10.6).
Fig 5 Effect of temperature on the activity of LipA The enz yme was
incubated in 50 m M phosphate buffer (pH 8.0) for 5 min at various
temperatures and activity ± SE was determined using p-NPP as
substrate The value obtained at 30 C was taken as 100%.
Trang 7compelling biochemical evidence for conservation of the
Ca2+-binding pocket in bacterial lipases and its importance
in enzyme stabilization
Effects of cations and inhibitors
LipA was incubated at 30C for 1 h with various cations
and inhibitors known to affect the Group I, II lipases,
reviewed by Gilbert [31] LipA showed an increase in
activity after exposure to low concentrations (1 mM) of
Ca2+, Mg2+, Co2+, Fe3+, and Rb+(Table 2) Increasing the metal concentration 10-fold had no further enhancing effect Zn2+(10 mM), Hg2+(1 mM) and Cu2+inhibited the lipase, reducing activity by 15%, 88%, and 85%, respect-ively Similar inhibition by heavy metals has been noted [29,32,38] Metal ions tested here may have variable affects
on lipase aggregation and on the substrate–water interface through interaction with free fatty acids [1,28] EDTA strongly inhibited enzyme activity; treatment with 1 mM resulted in 90% activity loss This effect was irreversible; i.e
no activity was recovered after incubation overnight at 4C with 20 mM CaCl2 Inhibition by EDTA probably results from its access to the Ca2+binding site and ion removal LipA was not affected by dithiothreitol (10 mM) or 2-mercaptoethanol (10 mM), suggesting a putative disulfide bridge is not required for activity Exposure to the serine-protease inhibitor phenylmethanesulfonyl fluoride did not result in significant inhibition Other serine hydrolases have shown similar resistance to such inhibitors in aqueous solutions [29]
Stability of the lipase in organic solvents Stability and activity in organic solvents are important characteristics of protein catalysts used in organic synthesis reactions Therefore, LipA stability in selected water miscible solvents was examined to assess this potential Lyophilized LipA, incubated with a variety of water-miscible organic solvents for 1 h at 30C, showed little effect, i.e ‡ 90% activity was retained (Table 3) Less than 30% of initial activity was lost after incubation with acetonitrile (30%), a solvent that has been shown to deactivate lipases in concentrations as low as 15% (v/v) [29,39,40] In contrast, lipase prepared from Chromobacterium viscosum showed high activity in acetonitrile where it is employed in trans-esterification reactions [41] Pyridine caused significant deactivation, i.e concentrations exceeding 15% (v/v) resul-ted in complete loss of activity within 1 h Similar sensitivities
to pyridine have been reported [27,39] Lyophilized LipA also did not show deactivation in the presence of nonpolar solvents in concentrations up to 99.8% (v/v) (data not shown) The lipase demonstrated very little loss in activity
Table 2 Effect of various inhibitors on LipA LipA was incubated with
various compounds that may inhibit the enzyme, and the remaining
activity was measured under standard conditions Enzyme preparations
were dialyzed against 30 m M Tris/HCl pH 8.0 prior to the experiment.
Lipase samples were incubated at 30 C for 1 h The remaining activity
(%) is expressed relative to the appropriate control value (with no
addition) post incubation Standard error for all experiments is less than
10% of the value reported ND, not determined.
Compound
Remaining activity (%) at
a concentration (mM) of:
a 20 m M CaCl 2 added post incubation with EDTA and incubated
at 4 C for 20 h.
Fig 7 Stabilizing effect of Ca2+on LipA Enzyme preparations were
dialyzed against 30 m M Tris/HCl (pH 8.0) prior to incubation in
30 m M Tris/HCl/2 m M MgCl 2 with (d) and without (s) 2 m M CaCl 2
Replicates (three) were examined at various times over a 30-h period
and activity plotted as percent of initial activity.
Table 3 Stability of LipA in selected organic solvents Lyophilized lipase was incubated with various organic solvents for 1 h at 30 C Residual activity was measured using standard conditions described in the text Results are expressed as the percentage of activity with no addition of solvent Standard error for all experiments is less than 5%
of the value reported.
Solvent
Remaining activity (%) at
a concentration (%, v/v) of:
Trang 8after exposure to solvents, indicating that it should be a useful
catalyst for organic solvent systems
Acinetobactersp RAG-1 lipase purified and
character-ized in this study demonstrated many properties
appropri-ate to a variety of industrial applications The enzyme is
stable for extended periods of time in the presence of
calcium LipA showed wide substrate specificity toward
medium and long chain esters of pNP Maximal reaction
temperature is 55C and LipA shows strong activity in the
presence of metals, inhibitors, and organic solvents Based
on these properties, we are investigating further the
potential of LipA to serve as a biocatalyst in selective
transesterification reactions
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