Vol.1, No.4, 79-90 (2013) Advances in Enzyme Research
Partial purification, immobilization and preliminary
biochemical characterization of lipases from
Rhizomucor pusillus
Ana Lúcia Ferrarezi1*, Daniele H. Pivetta2, Gustavo Orlando Bonilla-Rodriguez2,
Roberto da Silva2, José Manuel Guisan3, Eleni Gomes1, Benevides Costa Pessela4*
1Department of Biology, Laboratory of Applied Biochemistry and Microbiology, IBILCE-UNESP, São José do Rio Preto SP, Brazil;
*Corresponding Author: analu_fz@hotmail.com
2Department of Chemistry and Environmental Sciences, IBILCE-UNESP, São José do Rio Preto SP, Brazil
3Department of Enzyme Biocatalysis, Institute of Catalysis and Petrochemistry, CSIC, Campus UAM, Cantoblanco, Madrid, Spain
4Department of Biotechnology and Food Microbiology, Research Institute for Food Science, CIAL-CSIC, CalleNicolás Cabrera 9,
Campus UAM, Madrid, Spain; *Corresponding Author: b.pessela@csic.es
Received 24 June 2013; revised 14 August 2013; accepted 1 September 2013
Copyright © 2013 Ana Lúcia Ferrarezi et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lipases have important applications in bio-
technological processes, motivating us to pro-
duce, purify, immobilize and perform a bio-
chemical characterization of the lipase from
Rhizomucor pusillus. The fungus was cultivated
by solid state fermentation producing lipolytic
activity of about 0.5 U/mL(4U/g). A partial purifi-
cation by gel filtration chromatography in
Sephacryl S-100 allowed obtaining a yield of
about 85% and a purification factor of 5.7. Our
results revealed that the purified enzyme is very
stable with some significant differences in its
properties when compared to crude extract. The
crude enzyme extract has an optimum pH and
temperature of 7.5˚C and 40˚C, respectively. After
purification, a shift of the optimum pH from 7 to
8 was observed, as well as a rise in optimum-
temperature to 60˚C and an increase in stability.
The enzyme was immobilized on CNBr-Agarose
and Octyl-Agarose supports, having the highest
immobilization yield of 94% in the second resin.
The major advantage of immobilization in hy-
drophobic media such as Octyl is in its hyper
activation, which in this case was over 200%, a
very interesting finding. Another advantage of
this type of immobilization is the possibility of
using the derivatives in biotechnological appli-
cations, such as in oil enriched with omega-3 as
the results obtained in this study display the hy-
drolysis of 40% EPA and 7% DHA from sardine
oil, promising results compared to the literature.
Keywords: Enzyme Immobilization; Solid State
Fermentation; Purification; n-3 Polyunsatured F atty
Acids; Rhizomucor pusillus
Lipases are among the most used enzymes in organic
chemistry due to their high versatility [1-3], recogniz-
ing a broad range of substrates and being used in a very
wide range of reactions—from synthesis of structured
oils in food technology with fish oil enrichment of polyun-
satured fatty acids (PUFAs), to fine chemicals resolution
via hydrolytic reactions [4-7]. This diversity of reac-
tions makes lipase derivatives be employed in a wide
range of reaction media: from fully aqueous media or
water/co-solvent systems to anhydrous systems [1-3,5-8].
This makes the adsorbed lipases be exposed to very dif-
ferent medium conditions, and makes it very interesting
to compare different reversible immobilization approaches,
useful for each specific application. In this paper, we
study different strategies for immobilization by hydro-
phobic and covalent supports as well as characterization
of lipase from Rhizomucor pusillus.
Enzymes, due to their high selectivity, specificity and
activity under mild conditions may be a good option as
catalyst for a sustainable chemistry [4,9,10]. However,
they are biological catalysts and their features need to be
improved for most industrial applications [11-13]. One
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A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90
of these requirements is the convenience of preparing a
heterogeneous catalyst, that is, to have the enzyme in an
immobilized form [14-16]. Among the existing immobi-
lization strategies, the physical adsorption of the enzyme
on suitable supports is a very simple one, just by mixing
the enzyme and the support, the enzymes become rapidly
immobilized, usually with low impact on the enzyme
activity [17,18]. This produces a reversible immobiliza-
tion, and makes reuse of the supports possible after en-
zyme inactivation, thus, reducing the cost of the produc-
tion of the biocatalysts [17,18]. However, this reversibil-
ity may be a problem because the enzyme could desorb
from the support during operation, reducing the range of
conditions where the immobilized enzymes may be used.
Moreover, based on the particular mechanism of ac-
tion of lipases, there is one specific immobilization
strategy for lipases which is the use of hydrophobic sup-
ports. Lipases present two very different structures,
changing between a closed form, where the active centre
is secluded from the medium by an oligopeptide chain
called flap or lid, and an open form where this lid is
shifted and the active centre becomes accessible to the
substrate [19-21]. When in contact with hydrophobic
surfaces, such as oil drops, the open form of lipase is
adsorbed, involving the large hydrophobic surface
formed by the inner side of the lid and the active centre
[22,23]. Thus, in the specific case of lipases, one useful
immobilization technique is the interfacial activation of
the lipase by adsorption of the open form of the enzyme
on hydrophobic supports at low ionic strength [19-29].
This simple strategy allows the preparation of hyper-
activated lipase preparations, since the open form of the
enzyme is stabilized (at least when acting against hydro-
phobic substrates).
On the other hand, it has been described that the speci-
ficity and selectivity of lipases may be greatly altered by
the immobilization protocol, due to the conformational
changes that the lipases suffer during catalysis [22,23,28]
giving, as final result, an immobilized enzyme with very
different catalytic properties [14]. Therefore, it is impor-
tant to evaluate both the activity and stability of the im-
mobilized enzyme before selecting the optimal biocata-
2.1. Preparation of the Fungus by
Solid-State Fermentation (SSF)
R. pusillus was preserved on PDA slants. Inoculum
was by growing the fungus for 3 days at 45˚C. Solid state
fermentation (SSF) was performed in polypropylene
bags (21 cm × 12 cm) containing solid substrate (wheat
bran), a spore suspension of (5 × 105 colonies forming
units) and standard nutrient solution, containing 20 g·L1
peptone, 2 g·L1 of K2HPO4 and 0.5 g·L1 of MgSO4.
The crude enzyme extract was obtained by addition of 10
mL of distilled water per gram of fermented material,
stirred for 30 min, filtered, and centrifuged at 10,000 g,
at 6˚C. Finally, the supernatant was used as a crude en-
zyme solution for the preliminary biochemical charac-
terization and immobilization.
Samples were withdrawn every 24 hours for 5 days
and then their enzymatic activities and protein concen-
tration were determined.
2.2. Determination of Lipase Activity
These experiments were carried out by triplicate and
the standard error was under 5%.
2.2.1. p-Nitrophenylpalmitate (p-NPP)
A modified method of [30] was used for soluble and
partial pure enzyme. The synthetic substrate used was
p-nitrophenylpalmitate (p-NPP), and absorbance was
read in a Varian Cary 100 spectrophotometer at 410 nm
For the assay was used 0.05 M sodium phosphate
buffer (pH 7,0), incubation proceeded at 45˚C for 1 min,
and the reaction was stopped with 0.5 mL of a saturated
solution of sodium tetraborate (Na2B4O7). The molar
extinction coefficient of p-NP (e = 3.4 × 103 mol·L1)
was used to calculate the initial rate. One unit (U) of en-
zyme activity was defined as the amount of enzyme that
released 1 mmol of p-NP (p-nitrophenol) per minute. For
the partially purified enzymes the assays were performed
at the optimum conditions: pH 8.5 and 55˚C.
2.2.2. p-Nitrophenyl Butyrate (p-NPB)
The analysis of the activities of the soluble lipases and
their immobilized preparations were performed in a
quartz cuvette and analyzed spectrophotometrically by
measuring the increase in absorbance at 348 nm (extinct-
tion molar coefficient = 5150 M1·cm1) using 0.4
mMpNPB in 25 mM sodium phosphate buffer at pH 7,0
and 25˚C. Enzyme activity is given as μmols of p-NP
produced per minute per mg of enzyme (U) under the
conditions described earlier.
2.2.3. Biochemical Characterization of the Crude
Extract and Partially Pure Enzyme
1) Determination of the Optimum pH
The effect of pH on the enzyme activity was deter-
mined at 45˚C in suitable buffer and pH ranges of 3 - 10
(sodium citrate 0.05 M at pH 3, sodium acetate 0.05 M at
pH 4.0 - 5.0, sodium phosphate 0.05 M at pH 6.0 to 8.0
and glycine-NAOH 0.05 M at pH 9 - 10).
2) Determination of pH Stability
The effect of pH was tested at 45˚C, checking the en-
zyme activity every 30 minutes during 4 hours. The
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A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90 81
samples were kept in different buffers from pH 6.5, to
9.5 respectively. The enzyme assays were performed as
described earlier.
3) Determination of the Optimum Temperature
The optimum temperature of enzyme activity was de-
termined at temperature ranging from 25˚C to 70˚C in
sodium phosphate 0.05 M pH 7.5.
4) Determination of Thermal Stability of the Partially
Purified Lipases
The thermostability was measured by incubating 4 Ml
of enzyme extract at pH 8.5 and 55˚C, the optimum val-
ues for the partially purified enzymes. Samples were
withdrawn at chosen intervals, cooled and residual activ-
ity measured at optimum pH and temperatures.
2.3. Determination of Protein Concentration
Protein concentration was determined as proposed by
Bradford [31], using bovine serum albumin as a stan-
2.4. Gel Filtration Chromatography
A column (2.6 × 100 cm) from Pharmacia filled with
Sephacryl S-100 HR was attached to an FPLC (fast per-
formance liquid chromatography) Biologic System
(Bio-Rad). The column was equilibrated with 0.05 mM
phosphate buffer pH 7.5 containing 0.3 M NaCl. The
elution profile was monitored at 280 nm, collecting frac-
tions of 2 ml.
2.5. SDS-PAGE of the Free (Crude or Partial
Pure) and the Immobilized Enzyme
Proteins were analyzed by SDS-PAGE according to
Laemmli [32], using a precast 12% gradient gel (model
protean 16; Bio-Rad Laboratories, Richmond, Calif.).
Gels were silver-stained. To analyze amount of proteins
adsorbed on supports, a sample of the support was first
boiled in the presence of 1% SDS (w/v) and 2% 2-mer-
captoethanol (v/v) to desorb the proteins. Low molecular
weight markers from GE Healthcare were used (14 - 205
2.6. Zymogram Anal ys is
The zymogram was used to evaluate the presence of
isoenzymes. After the electrophoresis the gel was im-
mersed for 30 minutes in 0.05 M phosphate buffer pH
7.5 containing 2.5% Triton X-100 to remove SDS. The
gel was subsequently incubated in a mixture of two solu-
tions: Solution A: 1mL of acetone was dissolved in 40
mg of α-naphthyl acetate and 30 mg of β-naphthyl ace-
tate. Solution B: 120 mg of RR-fast-blue was dissolved
in 10 mL of isopropanol. Both solutions were mixed and
diluted with 0.1 M phosphate buffer pH 6.2 to 100 mL.
The gel was soaked in the solution mentioned earlier, and
incubated at 50˚C until the bands appeared [33].
2.7. Stability of the Crude Extract against
Different Storage Temperatures
Samples were kept in different environments: 80˚C,
20˚C and 4˚C for 30 days. Aliquots of these samples
were tested for enzymatic activity at 3 days intervals.
The experiments were carried out by triplicate and the
standard error was under 5%.
2.8. Effect of Ions on the Lipolytic Activity
To determine the effects of ions on the enzyme activity
20 mL of enzyme solution was mixed with different salt
solutions: CaCl2, MgCl2, NaCl, KCl, MnCl2 and ZnCl2 at
a final concentration of 2 mM. The enzyme activity as-
says were performed at the optimal conditions of pH and
temperature.The experiments were carried out by tripli-
cate and the standard error was under 5%.
2.9. Effect of the Fatty Acid Length on the
Lipolytic Activity
The standard substrate used in this study was p-nitro-
phenylpalmitate. However, tests were performed with the
following modifications: solution A consisted of each
substrate tested: p-nitrophenyl stearate, myristate p-ni-
trophenyl, p-nitrophenylcaprylate and p-nitrophenyl bu-
tyrate. Assays were performed in the optimal conditions
of activity. The experiments were carried out by triplicate
and the standard error was under 5%.
2.10. Enzyme Kinetics
Enzyme assays were performed with different concen-
trations (0.1 to 1.0 mM) of p-nitrophenylpalmitate, al-
lowing calculating the initial velocity Vo. The kinetic
constants Km and Vmax were calculated by nonlinear re-
gression using the Michaelis-Menten equation Vo =
(Vmax. [S])/(Km + [S]) as fitting model, using the soft-
ware QtiPlot version (ProIndepServ SRL, Ro-
mania). The experiments were carried out by triplicate
and the standard error was under 5%.
2.11. Immobilization Process of the
Isolipases from R. pusilllus
2.11.1. Octyl-Agarose Support
A volume of 10 mL of enzyme solution (7 mg total
protein/mL determined by Bradford’s assay [31]) were
mixed with 90 mL of 10 mM sodium phosphate at pH
7.0 and 4˚C. Ten grams of octyl-Sepharose previously
equilibrated with the immobilization buffer were added.
The supernatant and suspension activities were periodi-
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A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90
cally checked by the method described previously. After
the immobilization, the enzyme derivative was recovered
by filtration under vacuum. These adsorbed lipases were
used as biocatalyst in the resolution reactions.
To perform other studies, 1.5 mg proteins of enzyme
solution were mixed with1 g of octyl-Sepharose, in order
to prevent diffusion problems. All the other steps were
performed as in the standard preparations.
2.11.2. CNBr-Activated Sepharose Support
The immobilization of enzyme on CNBr-Sepharose
was performed for 15 minutes at 4˚C to reduce the possi-
bilities of a multipoint covalent attachment between the
enzyme and the support. Ten mL of prepared enzyme
solution were added to one gram of support and the reac-
tion was maintained for 40 minutes by constant stirring.
Periodically, activity of suspensions and supernatants
was measured by p-nitrophenyl butyrate as substrate.
The enzyme-support immobilization was ended by incu-
bating the support with 1 M ethanolamine at pH 8 for 2 h.
Finally, the immobilized preparation was washed several
times with 5 mM of sodium phosphate buffer pH 7 [28].
2.11.3. Immobilization on Glyoxyl-Agarose
Ten milliliters of enzyme solution were added to 100
mM sodium bicarbonate buffer at pH 10.5 and the pH of
the final solution was adjusted to pH 10.1. Then one
gram of glyoxyl-agarose (aldehyde activated support)
was added and the reaction was maintained for 24 h. Pe-
riodically, activity of suspensions and supernatants was
measured by using the pNPB assay. When the immobili-
zation was finished, 20 mg of NaBH4 were added during
15 min and then the suspension was filtered and washed
abundantly with distilled water (200 mL × 5). The im-
mobilization yield was >95%.
2.11.4. pH Stability of the Immobilized
Preparations of Octyl and CNBr activated support was
carried out with different buffers. Immobilized prepara-
tions were incubated in sodium acetate buffer 10 mM pH
5; Sodium phosphate buffer 10 mM pH 7 and Sodium
bicarbonate buffer 10 mM pH 10. The activity was
measured using the pNPB assay.
2.11.5. Thermal Inactivation of Different Lipase
Immobilized Prep arations
The different lipases preparations (0.5 mg/g) were in-
cubated in 10 mM sodium phosphate at pH 7.0 and 45˚C
- 55˚C. Samples were withdrawn intermittently and their
activities were measured using the pNPB assay in the
interval 1 - 24 hours. The experiments were carried out
by triplicate and error was never over 5%.
2.12. Analysis of Polyunsaturated Free Fatty
Acids (PUFA) by HPLC–UV
The immobilized lipases were submitted to sardine oil
hydrolysis to obtain PUFAs. After a given time, aliquots
of 0.1 mL of organic phase were withdrawn and dis-
solved in 0.8 mL of acetonitrile. The organic phase was
easily separated (in less than 5 min) from the aqueous
phase when stirring of the biphasic system was stopped.
The unsaturated fatty acids produced were analyzed by
RP-HPLC [Spectra Physic SP 100 coupled with an UV
detector Spectra Physic SP 8450 (Spectra Physics, Santa
Clara, CA. USA)] using a Kromasil C8 (15 cm 9 0.4 cm)
column. Products were eluted at a ow rate of 1.0
mL/min using acetonitrile-10 mMTris–OH buffer at pH 3
(70:30, v/v) and UV detection performed at 215 nm. The
retention times for the unsaturated fatty acidswere: 9.4
min for EPA and 13.5 min for DHA. These enzymatically
produced PUFA were compared to their corresponding
pure commercial standards.
3.1. Lipase Production
Maximum enzyme activity was observed at 72 h, as
shown in Figure 1, and 0.5 U/ml equivalent to 4.1 U/g
enzyme activity was obtained during enzyme production.
Maximum lipolytic activity was reached at the third
day, decreasing afterwards. This is likely due to the
presence of a protease. Another explanation for the dropin
enzyme activity could be an inhibitory effect of other
metabolites secreted in the course of the fermentation.
3.2. Partial Purification of Lipases Produced
by the Thermophilic Fungus R. pusillus
The lipases present in the extract were partially puri-
fied through gel filtration chromatography (Figure 2),
showing at least two enzymes, as deduced from the ac-
tivity profile. After pooling the fractions with highest
activity, we found an 84.9% yield and a 5.7 purification
factor (Ta b l e 1 ). These results can be considered prom-
ising when compared to previous works (results not
shown). However, when we tried to increase the degree
of purification by ion exchange chromatography
(DEAE-Sepharose) or hydrophobic interaction (Octyl-
Sepharose) the activity decreased significantly. Thus, the
characterization experiments were performed using the
isolipases partially pure.
When these data are compared to those reported for
the lipase from Aero monascaviaeAU04, a mesophilic
bacterium by submerged fermentation [34], the authors
obtained a total recovery of 28.76% with a purification
factor of 3.4 using phenyl sepharose. After purification
by ion exchange chromatography with DEAE-Sepharose,
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A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90 83
Figure 1. Hydrolytic activity of the crude extract obtained by-
solid fermentation of the thermophilic fungus Rhizomucor pu-
sillus. The experiments were performed in triplicateand the
standard error was under 5%.
Figure 2. Elution profile of a gel filtration chromatography
performed on Sephacryl S-100 support. Conditions: 0.05 M
sodium phosphate buffer pH 7.5 containing 0.3 M NaCl. Sym-
bols: () protein profile detected at 280 nm; () lipolytic ac-
tivity profile in U/mL.
Table 1. Partial purification of lipases produced by Rhizomu-
Step Volume
(%) Purification
factor (fold)
extract 15 128.3 13.3 9.6 100.0 1.0
filtration 13.5 108.9 2.00 55.0 84.9 5.7
Pastore et al. [35] obtained a 0.4% yield and purification
factor of 3.9 for lipases from Rhizopus sp. In the same
study, the authors reported 0.4% of recovery and purifi-
cation factor of 6.9 using the hydrophobic resin phenyl-
Liu et al. [36] also purified a microbial lipase; they
used gel filtration with Sephadex G-75, obtaining a
12.6% yield and a purification factor of 3. After applying
to a DEAE-Sepharose column (ion exchange chroma-
tography) obtained 4.8%, of yield and a purification fac-
tor of 4.8 fold.
The analysis of the protein profile by SDS-PAGE
shows that there are many contaminants remaining after
gel filtration (results not shown). This can be caused by
the formation of high molecular weight aggregates,
commonly reported in studies of purification of lipases,
which can hinder the process [37]. The formation of
these aggregates may be due to the presence of lipids or
the hydrophobic characteristic of the protein structure.
The zymogram profile (results not shown) showed two
spots for lipases that would have molecular weights be-
tween 44 and 55 kDa. The lipase displaying higher mo-
lecular weight (the first peak at the gel filtration profile,
Figure 2) displayed higher activity.
3.3. Biochemical Characterization of the
Crude Enzymatic Extract and the
Partially Purified Lipases
The lipolytic activity profile as a function of pH (Fig-
ure 3(a)) displayed a peak at pH 7.5 with enzyme ac-
tiveity. The lipolytic activity of the crude extract was the
sameor higher than 70% in the pH range from 7 to 10,
whereas for the partially purified extract the highest ac-
tivity was recorded at pH 8.5, in agreement with opti-
mum pH values in the literature for lipases, which tend to
be alkaline.
This feature is valuable for their potential biotechno-
logical applications, because these lipases could be ef-
fective in an alkaline medium, for example in cleaning
solutions [38]. It may be noticed that the mixture of these
lipases has activity in a wide range of pH values; be-
tween 5 and 9 displays activity above 75%, a feature that
suggests a potential application of these enzymes in a
range of various reaction conditions.
The difference between the optimum pH of the en-
zyme found for the crude extract (pH 7.5) and the par-
tially purified enzymes could be attributed to contami-
nants that were removed and would interfere in the activ-
ity of these enzymes.
The activity profile as a function of temperature (Fig-
ure 3(b)) showed that the crude extract had an optimum
temperature of 40˚C, and that for the partially pure lipase
exhibited a higher value: 55˚C. It is evident that there
was an increase in optimum temperature of the partially
pure lipase compared to the crude extract. This could be
due to the elimination of proteins and proteases that can
nhibit the activity of these lipases or denature at lower i
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A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90
Copyright © 2013 SciRes.
Figure 3. (a) Effect of pH on the lipolytic activity; (b) Effect of the temperature on the lipolytic activity: Symbols: () The
crude enzymatic extract; () partially purified lipases. (c) pH Stability of the partially purified lipase maintained at 45˚C for
4 hours. Conditions: pH 8.5 and 55˚C. Symbols: () pH 9.5; () pH 8.5; () pH 7.5; () pH 6.5. (d) Thermal stability of
the partially pure lipases from R. pusillus maintained at different temperatures at pH 8.5 for up to 4 hours. Conditions: pH
8.5 and 55˚C. Symbols: () 4˚C; () 25˚C; () 35˚C; () 45˚C; () 55˚C; () 65˚C.
temperatures promoting aggregation with the enzymes.
At temperatures below 40˚C, their activity was low,
probably due to the high intrinsic rigidity of thermophilic
proteins. These proteins demand high temperature of ac-
tivity (above 40˚C) to promote the thermal motion and
increased flexibility essential for catalytic activity [39].
Studies with intracellular lipases from athermophilic
Rhizomucor miehei [40], obtained an optimum pH and
temperature of 7.0 and 40˚C respectively. Another lipase
which has similar characteristics produced by a meso-
philic Rhizopus oryzae [41] showed optimal conditions
of activity at pH 7.5 and 35˚C. Liu et al. [36] also ob-
tained similar results for the lipase from a mesophilic
Aureobasidium pullulans HN2.3, which exhibited opti-
mum temperature at 35˚C and pH 8.5.
Velu et al. [34] studied lipases of Aeromonas caviae
and obtained as optimum pH 7.0 and 60˚C. Studying a
lipase from a thermophilic Bacillus sp. PTI-001, Nawani,
Kaur. [42] found pH 8 and temperature 60˚C as optimum
Figure 3(c) shows that the lipases partially purified of
R. pusillus incubated at different pH were less stable at
more alkaline pH values (8.5 and 9.5) after 150 minutes,
with steeper decline than for the samples kept at pH 6.5
and 7.5. Most fungal lipases present maximal activity in
a temperature range of 30˚C to 65˚C. Generally, they
have optimal activity at pH of (4.5 - 6.0). Furthermore,
the enzyme activity is also affected by pH, which affects
the enzyme charge distribution and, consequently, the
substrate binding and its catalysis.
The thermal stability of these enzymes (Figure 3(d))
is greater when kept at 4˚C, but the enzymatic activity of
these lipases remains constant and close to 100% when
kept at temperatures up to 45˚C.
The lipase maintained at 55˚C showed more than 80%
of its activity within 60 minutes of incubation. After 240
minutes, the enzyme still showed approximately 40% of
its residual activity.
A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90 85
However, when kept at 65˚C, the lipase activity
dropped to 40% by 60 minutes, which was soon lost,
leaving only about 25% activity at the end of 240 min-
3.4. Stability of the Lipases from the Crude
Extract at Different Storage Conditions
In order to determine the best storage condition of
these crude enzymes, we analyzed the effect of time on
enzyme activity for samples stored at different tempera-
tures. When kept frozen at 80˚C or 20˚C, the enzyme
displayed a slight loss of activity, but if stored at 4˚C, it
lost about more than 30% of its initial activity after 30
3.5. Effect of Cations on the Lipolytic
As is shown in Table 2, most of the tested cations in-
creased activity; the addition of Mg2+ to the enzyme so-
lution, 62.1% of its activity was increased on the lipase
partially purified from Rhizomucor pusillus, which is
slightly higher than the activity obtained in the presence
of Na+. Ca2+ and Mn2+ also significantly activated the
activity of these lipases, while K+ did not practically af-
fect its activity. However, when Zn2+ was added, there
was a significant inhibition of enzyme activity.
Sidhu et al. [43] observed that the lipase from Bacil-
lus sp. with optimum temperature of 50˚C and pH 8 was
also activated in the presence of Ca2+ and Na+. Chartrain
et al. [44] observed that extracellular lipases of P.
aeruginosa were activated by Ca2+ (1.26-fold) and
strongly inhibited by Zn2+ (94%). Sharon et al. [45]
showed that the activity of lipases from P. aeruginosa
KKA-5 also increased in the presence of Ca2+and Mg2+,
but were inhibited by Mn2+.
Table 2. Effect of ions (2 mM final concentration) in the enzy-
matic activity of lipases partially purified from R. pusillus.
Assays were performed in triplicate and analyzed in the optimal
conditions of the enzyme (pH 8.5 and 55˚C).
Cation Relative activity % (arithmetic
mean ± standard deviation)
CONTROL 100.0 ± 0.11
CaCl2 154.6 ± 0.35
MgCl2 162.1 ± 0.26
NaCl 159.9 ± 0.16
KCl 106.2 ± 0.27
ZnCl2 31.9 ± 0.6
MnCl2 132.8 ± 0.21
Many of known enzymes require the presence of metal
ions for catalytic activity. These lipases appear to belong
to this group (metalloenzymes), behaving as metal-acti-
vated enzymes, which they associate weakly to metal
ions from solution [46]. In this case, the ions play a
structural function, rather than catalytic, since even in the
absence of metals, these lipases exhibit high enzyme
3.6. Effect of the Fatty Acid Chain Length on
the Activity of the Partially Purified
Tests with different substrates (Table 3) showed that
the lipases from Rhizomucor pusillus have more activity
with the substrate p-nitrophenylmyristate (C14:0), and
decreasing activity with palmitate (C16:0) > caprylate
(C8:0) > stearate (C18:0). That is, for the fatty acids
tested (all saturated) showed a preference for carbon
chains longer than eight carbons, since for p-nitrophenyl
butyrate (C4:0) the activity was around 25% compared
with p-nitrophenyl-myristate.
3.7. Kinetics Parameters of the Enzyme
From the values of enzyme activity (initial velocity)
with different substrate concentrations of p-nytrophenyl-
palmitate (Figure 4), we estimated the kinetic constants,
Vmax and Km (Michaelis-Menten constant). The values
obtained by nonlinear regression were Vmax = 12.6 ±
0.5 mmoles/min and Km = 0.2 ± 0.0 mM. It is important
to stress that the calculated values for the lipases from R.
pusillus are apparent, since they are a result of the action
of two enzymes.
Comparing these values to other kinetic analyses using
the same substrate, Nawani, Kaur [42] analyzed lipases
from thermophilic Bacillus sp, finding a V max = 0.032
µmoles/mL and Km = 0.19 mM. Brabcová et al. [47]
found a higher Km for the same substrate for an ex-
tracellular lipase from the mesophilic fungus Geotrichum
candidum 4013: 0.406 mM.
Table 3. Affinity of the partially pure lipase in relation to dif-
ferent synthetic substrates at 0.8 mM final concentration. As-
says were performed in optimal conditions of the enzyme for
p-NPP (palmitate), pH 8.5 and 55˚C.
(p-Nitrophenyl-fatty acid) Relative activity (%)
Myristate 100.0 ± 0.79
Palmitate 91.4 ± 0.54
Caprate 81.5 ± 0.86
Stearate 78.2 ± 1.34
Butyrate 24.7 ± 1.00
Copyright © 2013 SciRes. OPEN ACCESS
A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90
Figure 4. Enzyme Kinetics performed with different concentra-
tions of p-nitrophenyl-palmitate in the optimal conditions of
enzymatic activity: pH 8.5 and 55˚C.
3.8. Immobilization of Crude Lipases on
Octyl-Agarose and CNBr Activated
Table 4 shows the immobilization course of the crude
enzyme octyl-Sepharose and CnBr supports. In all cases,
immobilization was very rapid (full immobilization was
achieved after only 15 minutes on octyl-Sepharose).
However, the effect on enzyme activity was very dif-
ferent. Immobilization on the CNBr did not produce any
positive or negative effect on the enzyme activity, while
immobilization on octyl-Sepharose increased the enzyme
activity by a 10 fold factor. This enhancement in the li-
pase activity by immobilization on hydrophobic supports
hasbeen described previously for many lipases, and it is
correlated to its interfacial activation [28,29].
On octyl-Sepharose was achieved almost complete en-
zyme immobilization and 100% of the initial enzyme
activity was expressed on the support, leaving only 25%
- 30% of the proteins. In contrast, using the CNBr sup-
port only 30% of activity was immobilized. In fact, the
SDS-PAGE analysis of the proteins adsorbed on the
supports showed that all proteins have been immobilized
in a similar way, while octyl-Sepharose selectively ad-
sorbed about only 50% of the proteins (Data not shown).
Thus, immobilization on octyl-Sepharose permitted
the one step immobilization, purification and hyperacti-
vation of enzymes preparation, while CNBr is also an
efficient method of enzyme immobilization, but neither
purify nor hyper activate the lipase.
3.8.1. Stability of the Immobilized Preparations
The stability of the derivatives and the soluble enzyme
was tested at room temperature and pH 5. In Figure 5(a),
we see that both the soluble enzyme and the derivative
Ta b le 4 . Immobilization yield and expressed activity of R. pu-
sillus lipase on two different supports.
Immobilization supports Immobilization
yield (%) Expressed
activity (%)
CNBr-activated support 30 80
Octyl-agarose support 95 237
prepared on octyl have the same stability profile. Both
have some hyperactivation, while CNBr have a com-
pletely different behavior.
The question remains in the behavior of CNBr deriva-
tive, since within the first 5 hours, the activity falls to
almost 50% at pH 5, when the derivative had a higher
enzyme expression.
Regarding pH 7 (Figure 5(b)), both the soluble en-
zyme and derivatives, have a completely different be-
havior. Here, the two derivatives (CNBr and Octyl) have
a similar behavior, while the soluble enzyme activity
remained almost unchanged, after 24 hours its activity
drops by 25%.
At pH 10, the derivative activity in CNBr, drops
sharply and the curious case is presented with the soluble
enzyme. As they are not completely pure, so that the
protein-protein interaction confers some stability and
hyperactivation, at some point, unlike the derivative oc-
tylagarose, Figure 5(c).
3.8.2. Thermal Stability of the Immobilized
Another interesting feature of an immobilization is the
study of the effect of temperature on immobilized enzyme.
Figures 6(a) and (b) show the inactivation course of the
lipase immobilized on CnBr and octyl-agarose, stability
at 45˚C and 50˚C was compared. CNBr-Sepharose has a
mild covalent attachment –having quite similar stability
to the soluble enzyme without any intermolecular inter-
actions (e.g., lipase-lipase interactions). Hence, it is in-
triguing that the CNBr derivative activity and hyper ac-
tivity were intact and maintained about 180% of its ini-
tial activity when analyzed at 45˚C.
While the octyl-agarose derivative, which was ex-
pected to exhibit this behaviour did not, but rather the
hydrophobic and expressed enzyme activity was much
higher at the beginning, as shown in Figures 6(a) and
3.9. Hydrolysis of Sardine Oil Using
Immobilized Lipase on Octyl and
Using octyl-Sepharose derivatives of lipases the hy-
drolysis of sardine oil under standard reaction conditions
was studied, in addition to the HPLC–UV analysis to
follow the rate of PUFA release and the EPA/ DHA se-
Copyright © 2013 SciRes. OPEN ACCESS
A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90
Copyright © 2013 SciRes.
Figure 5. (a) Stability of crude extract and immobilized lipase in pH 5; (b) Stability of lipase in the crude extract and
immobilized lipase at pH 7; (c) Stability of lipase in the crude extract and immobilized lipase at pH 10. Symbols: ()
Crude extract of soluble lipase; () Lipase adsorbed on CNBr-activated; () Lipase adsorbed on octyl-agarose.
Figure 6. (a)Thermostability of CNBr derivative at pH 7. The activity was measured as was described in materials
and methods. (b) Thermostability of the Octyl derivative at pH 7; Symbols: () 45˚C; () 50˚C.
lectivity. HPLC-light scattering analysis was also carried
out to determine lipase selectivity towards polyunsatu-
rated fatty acids:eicosapentaenoic acid (EPA) (20-5, n-3;
polyunsaturated fatty acid d) and docosahexanoic acid
(DHA) (22-6, n-3; polyunsaturated fatty acid). EPA and
DHA are widely available in fishoil and are recognized
A. L. Ferrarezi et al. / Advances in Enzyme Research 1 (2013) 79-90
to have beneficial health effects. In this way, the validity
of the release of PUFA for the accurate measurement of
lipolytic activity of lipases could be determined.
As a routine our group has developed new methodolo-
gies for the analysis of both PUFAs(EPA and DHA).
Enabling quick analysis of the hydrolysis of these com-
pound with the derivatives, just to see, how they were or
not selective in such reactions. Based on this we ana-
lyzed only at pH 7 and different temperatures. As shown
in Table 5, the octyl derivative achieved the highest
yields of EPA and DHA at 37˚C, with selectivity close to
Morrissey and Okada [48] reported the hydrolysis of
33% of EPA and 29% of DHA from sardine oil after 9 h
of reaction using the commercial lipase of Candida
rugosa (not immobilized) at a concentration of 500 U. It
is worth mentioning that there is no significant selectiv-
ity difference between these two fatty acids, differently
from what we observed in this study, with a lipase more
selective for EPA. The reaction reported by those authors
was faster; however, it took 500 U for hydrolysis,
whereas in this work only 130 U were able to carry out
the hydrolysis of 40% and 7% of EPA and DHA respec-
tively. These promising results have allowed us to work
on improving the production of these lipases and search
for new and better methods of mobilization for future
The enzyme was produced with a very good activity
level, subsequently with simple purification processes,
even without the separation of the isolipases, and yield
close to 90% was obtained.
The expression of the enzyme immobilization map-
ping is excellent, due to immobilization on octyl-agarose,
which reached hyperactivation levels in the order of
200%, which is excellent for studies of enantioselectivity
against filing details.
Furthermore, the derivatives stability versus tempera-
ture and pH were excellently allowing use in industry
where conditions may be somewhat drastic. The method
used of enzyme immobilization applied was very simple
Table 5. Yield of EPA and DHA in 72 hours reaction of hy-
drolysis of fish oil and selectivity for these fatty acids. Condi-
tions: Fish oil in Tris 10 mM pH 7 buffer and cyclohexane-
under magnetic agitation.
Derivative EPA
(20:5 n-3)(%) DHA
(22:6 n-3)(%) Selectivity
Octyl-agarose pH 7, 25˚C 11.2 4.2 2.7
Octyl-agarose pH 7, 45˚C 10.0 2.5 4.0
Octyl-agarose pH 7, 37˚C 40.0 6.8 5.9
and very easy. All amount of the soluble enzyme offered
before on each support was completely and quickly ad-
sorbed in all cases with excellent yields on immobiliza-
The lipases from R. pusillus showed important charac-
teristics with potential for industrial applications; in par-
ticular, they displayed promising results in the enrich-
ment of fish oil with PUFA omega 3.
The authors are grateful to Capes-Carolina Foundation (Brazil) for
the scholarship of post-doctoral (A.L. Ferrarezi), held at the Instituto de
Investigaciones en Ciencias de los alimentos, CIAL-CSIC, Madrid,
Spain. The author is thankful to FAPESP and CNPq (Brazil) for finan-
cial support and Consolider INGENIO 2010 CSD2007-00063 FUNC-
FOOD (CICYT), the Spanish Ministry of Science and Innovation.
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