Turk J Biol
35 (2011) 35-44
© TÜBİTAK
doi:10.3906/biy-0902-14
The effects of the composition of growth medium and
fermentation conditions on the production of
lipase by R. delemar
Ünsal AÇIKEL1,*, Mehtap ERŞAN1, Yeşim SAĞ AÇIKEL2
1
Department of Chemical Engineering, Cumhuriyet University, 58140, Campus, Sivas - TURKEY
2Department of Chemical Engineering, Hacettepe University, 06800 Beytepe, Ankara - TURKEY
Received: 10.02.2009
Abstract: Lipases (triacylglycerol hydrolases) are hydrolytic enzymes that can catalyze the hydrolysis of the ester bond
of long-chain acylglycerols at the oil-water interface. The present study investigated the effects of inducers, surface-active
materials, activators, and inhibitors in the fermentation medium on lipase activity in Rhizopus delemar. In the presence
of certain commercial oils and tributyrin as an inducer, lipase activity decreased in the order of sunflower oil > soybean
oil > hazelnut oil > corn oil > tributyrin > olive oil. In addition, the effects of the stirring and aeration rates on lipase
activity were investigated. To investigate the effect of surface-active materials on lipase activity 2 different surface-active
materials, Tween-80 and rhamnolipid (biosurfactant), were used as comparatives with tributyrin. Maximum lipase activity
was observed in the fermentation medium containing Tween-80 as both an inducer and surface-active material. The
combined effects of molasses and sucrose on lipase production by R. delemar were also investigated. Lipase activity in
the presence of activators decreased in the order of NaCl > KCl > CaCl2 > MgCl2 > gum arabic > EDTA, and maximum
lipase activity of 964.55 U/L was obtained. The presence of heavy metal ions in the fermentation media severely inhibited
2+
2+
2+
2+
lipase activity. Lipase activity in the presence of heavy metal ions decreased in the order of Fe > Mn > Co > Ni .
Key words: Lipase production, R. delemar, fermentation, batch stirred reactor
Üreme ortamı bileşimi ve fermentasyon koşullarının R. delemar’ın
lipaz üretimi üzerine etkileri
Özet: Lipazlar (triaçilgliserol hidrolazlar), yağ-su arayüzeyinde, uzun zincirli açilgliserollerin, ester bağlarının hidrolizini
katalizleyen hidrolitik enzimlerdir. Bu çalışmada, Rhizopus delemar’ın lipaz aktivitesi üzerine, fermentasyon ortamındaki
indükleyicilerin, yüzey aktif maddelerin, aktivatörler ve inhibitörlerin etkileri araştırıldı. İndükleyici olarak, belirli ticari
yağlar ve tribütirinin varlığında, elde edilen lipaz aktiviteleri, sırasıyla ayçiçeği yağı > soya yağı > fındık yağı > mısır yağı
> tribütirin > zeytin yağı olarak azalmaktadır. Buna ek olarak, karıştırma hızı ve havalandırma hızının lipaz aktivitesi
üzerine etkileri araştırıldı. Yüzey aktif maddelerin lipaz aktivitesine etkisinin incelenmesi için, iki farklı yüzey aktif
madde, Tween-80 ve rhamnolipid (biyosurfaktan), tribütirin ile karşılaştırmalı olarak kullanılmıştır. Maksimum lipaz
aktivitesi, hem bir indükleyici hem de bir yüzey aktif madde olarak Tween-80 içeren fermantasyon ortamında elde
edilmiştir. R. delemar’ la lipaz üretimi üzerine, melas ve sakkarozun birleştirilmiş etkileri de araştırılmıştır. Aktivatörlerin
varlığında elde edilen lipaz aktiviteleri, sırasıyla NaCl > KCl > CaCl2 > MgCl2 > arap zamkı > EDTA olarak azalmış ve
maksimum lipaz aktivitesi 964,55 U/L olarak bulunmuştur. Fermantasyon ortamında bulunan ağır metal iyonları lipaz
+2
+2
aktivitesini şiddetli bir şekilde düşürmüştür. Ağır metal iyonlarının varlığında lipaz aktivitesi sırasıyla Fe >Mn
+2
+2
>Co >Ni olarak azalmıştır.
Anahtar sözcükler: Lipaz üretimi, R. delemar, fermentasyon, kesikli karıştırmalı reaktör
35
The effects of the composition of growth medium and fermentation conditions on the production of lipase by R. delemar
Introduction
Lipases (triacylglycerol acyl hydrolases) are one of
the most important classes of industrial enzymes.
Lipases are biological catalysts that hydrolyze
triglycerides to di- and monoglycerides, glycerol, and
fatty acids, and under certain conditions catalyze the
reverse reaction, forming glycerides from glycerol and
fatty acids (1). Worldwide, there is considerable
interest in the use of commercially available lipases
for the hydrolysis of oils, synthesis of esters,
interesterification of oils and fats, synthesis of
peptides, and resolution of racemic mixtures to
produce optically active compounds (2-6). This
interest stems from such characteristic lipase
properties
as
substrate
specificity
and
stereospecificity, and their ability to catalyze
heterogeneous reactions at the interface of watersoluble and water-insoluble systems (7-9).
Microbial lipases have a broad spectrum of
industrial application, as they are more stable and
suitable for genetic manipulation than plant and
animal lipases, and they can be obtained cheaply.
Microbial lipases are used in the food industry (10),
detergents (11), pharmaceuticals (12), cosmetics (13),
leather processing (6), the paper industry (5),
biosensors (14), biodegradation of polymers (15),
biodiesel-fuel production (16), the manufacturing of
fine chemicals (in particular, pesticides) (17), and
waste management (18,19).
The lipase from Rhizopus delemar is currently the
subject of intensive research and industrial interest
because it produces at least 3 extracellular lipases,
each of which exhibits a strong preference for the
hydrolysis of ester bonds at the sn-1 and sn-3 positions
of triacylglycerol. Partially purified mixtures of these
enzymes are used in a variety of reactions, including
ester and glyceride synthesis, glyceride restructuring,
and the exchange of the acyl groups of phospholipids
(20-22). Regulation of the synthesis of R. delemar
lipase is complex, and the effects of fermentation
conditions, such as pH, temperature, inoculum level,
carbon and nitrogen sources, inducers, and activators,
can be major (23). Data on the fermentation process
are critical to the search for optimal operation
conditions for enzyme production (24).
present study examined this process in relation to the
composition of the growth medium, including
selection of the optimum inducer or surfactant, and
fermentation conditions, such as the optimum
quantity of inducer and surfactants, and the stirring
and aeration rates.
Materials and methods
Microorganism and growth medium
R. delemar was obtained from the US Department
of Agriculture Culture Collection (NRRL 2872). R.
delemar was grown in an agitated liquid-basal
medium that contained the following ingredients (g
L-1): molasses sucrose: 1; K2HPO4: 0.5; KH2PO4: 0.5;
MgSO4.7H2O: 0.2; yeast extract: 2. For the
experiments performed with molasses sucrose the
growth medium included molasses diluted to the
desired sucrose concentration as the main carbon
source. Molasses was obtained from Ankara Sugar
Factory (Turkey). Molasses is a co-product of sugar
production from sugar beet, and is defined as the
runoff syrup from the final stage of crystallization.
Sugar beet molasses is a solution of sugar, and organic
and inorganic matter in water, with a solid substance
of 84% (w/w). Total sugars (mainly sucrose) constitute
approximately 50% (w/w) of molasses, ash 11% (w/w),
and total nitrogen-containing compounds (mainly
betaine) 7%-8% (w/w) (Table). Molasses also contains
biotin, and to a lesser extent invert sugar and
raffinose, trace elements such as K+, Na+, Ca2+, Mg2+,
Fe2+, Al+, Cl-, SO4-, PO4-, and NO3-, metal oxides, and
acid anhydrates.
Table. Organic sugar and N2 content of molasses obtained from
Ankara Sugar Factory.
Sa
Pb
Qc
Invert sugar
Betain
Total N2
Raffinose
Density
a
The optimum conditions for lipase production by
R. delemar have yet to be described in full detail. The
36
% 83.92
% 50.03
% 59.62
% 0.406-0.500
% 5.0 -6.0
% 2.0
% 0.8 – 1.0
1.3
Amount of solid substance
Polarization
c
P/S
b
Ü. AÇIKEL, M. ERŞAN, Y. SAĞ AÇIKEL
Analytical procedure
Dry weight of the biomass was determined by
filtering the sample medium over filter paper. The
filters were dried at 60 °C until constant weight was
achieved. For lipase activity measurement the
spectrophotometric method of p-nitro-phenylpalmitate (pNPP) was used as follows: solution A
contained pNPP dissolved in propan-2-ol; solution B
contained gum arabic and Triton X-100 dissolved in
distilled water, and the pH of the solution was
adjusted to 8.0. The substrate solution was prepared
by adding solution A to solution B dropwise under
intensive stirring. The pH of the substrate solution
was adjusted to 8.5 using Tris buffer. For lipase activity
measurement an enzyme sample was added to the
substrate solution. The reaction mixture was
incubated at 37 °C for 30 min in a rotary shaker
operating at 150 rpm. The rate of p-nitrophenol
liberation was measured at 410 nm using a
spectrophotometer. One enzyme unit (U) of lipase
activity was defined as the amount of enzyme needed
to liberate 1 μmol of pNP per minute at the conditions
described for each assay system (25).
production, and facilitate its purification and
utilization. The production of microbial lipases is
generally induced by fat-related carbon sources. The
effect on the lipase enzyme activity of R. delemar of
certain commercial oils and tributyrin as inducers was
investigated in fermentation media containing
molasses sucrose as the main carbon source. The oils
investigated included sunflower oil, soybean oil,
hazelnut oil, olive oil, and corn oil. Inducers were
added at 0.5% (v/v) to the fermentation media.
Maximum enzyme activity was observed in the
fermentation medium containing sunflower oil
(Figure 1). The inclusion of sunflower oil in the
fermentation medium resulted in a significant
increase in lipase production. In the presence of
sunflower oil maximum lipase activity was 93.07 U/L,
which was approximately 19.7-fold greater than that
in the absence of an inducer. The production of
extracellular lipase increased steadily with cultivation
time. Maximal enzyme activity was reached after 5
days of culturing. Most of the lipase was produced by
R. delemar during the late exponential phase of
growth. When other commercial oils were used
together with molasses, a significant increase in lipase
activity was also observed. Lipase activity decreased
in the order of sunflower oil > soybean oil > hazelnut
oil > corn oil > tributyrin > olive oil. R. delemar lipase
exhibits a strong preference for the hydrolysis of ester
bonds at the sn-1 and sn-3 positions of triacylglycerol.
100
60
40
20
0
Results and discussion
The effect on lipase activity of certain
commercial oils and tributyrin as inducers
The addition of lipase inducers to the culture
medium of microorganisms can enhance lipase
Sunflower oil
Soybean oil
Hazelnut oil
Olive oil
Corn oil
Tween- 80
Rhamnolipid
Tributyrin
80
Activity (U/L)
Inducers or surfactants were added to the culture
broth in concentrations of 0.5%-4% (v/v). Inoculation
was carried out aseptically with the cells at the end of
the lag phase. The optimum inoculum ratio (volume
of inoculum/production volume of the bioreactor)
was determined to be 5/1000, and all inoculum was
added to the fermentation media at this ratio. Flasks
(250 mL) containing 100 mL of the growth liquid
medium were incubated on a rotary shaker at 100 rpm
at 30 °C. The pH of the medium was initially adjusted
to 8.0, determined as optimum for lipase production
in our previous studies, and allowed to follow its
natural course. In order to investigate the effect of the
stirring and aeration rates, lipase fermentation was
performed in a batch-baffled and magnetically stirred
reactor. The working volume of the reactor was 100
mL.
0
2
4
6
8
Time (Day)
10
12
Figure 1. Time course of lipase activity in fermentation media
containing various oils, surfactants, and tributyrin as
inducer (pH = 8.0; temperature = 30 °C; concentration
of molasses sucrose = 1 g/L, concentration of inducer =
0.5% (v/v)).
37
With this marked positional selectivity, R. delemar
lipase exhibits little selectivity for hydrolysis of
substrates with different fatty acid moieties. Studies
on triglyceride mixtures show that tributyrin is the
least preferred substrate (22).
Li et al. reported that oleic acid was the best
inducer for enhancing the production of Rhizopus
arrhizus lipase, and the highest lipase activity
obtained was 3.62-fold greater than that before oleic
acid feeding (26). Elibol and Özer reported that in the
presence of corn oil lipase activity of immobilized R.
arrhizus was approximately 2.5-fold greater than that
in the absence of an inducer (27). Rodriguez et al.
used different triglycerides (olive, sunflower, corn,
peanut, walnut, and grape seed oils) for Rhizopus
homothallicus lipase production and interestingly
observed that this fungal strain was able to produce
similarly high lipase activity with each oil (28).
As can be seen, reported results concerning the
best inducer for the production enhancement of
Rhizopus sp. lipase vary. The results obtained in the
present study are in agreement with those for R.
delemar lipase production reported by Espinosa et al.
(29). Different oils were tested as the sole carbon
source. Sunflower oil yielded the highest enzyme
production, and tributyrin and olive oil yields did not
significantly differ, and were generally low.
The effect of the sunflower oil concentration on
maximum lipase activity was also investigated at 4
different concentrations, varying from 0.5%-4% (v/v).
Lipase activity decreased as the sunflower oil
concentration increased; the 0.5% (v/v) sunflower oil
concentration was the optimum inducer
concentration.
Unlike lipase activity, the biomass concentration
increased as the sunflower oil concentration increased
up to 1% (v/v), and then the maximum biomass
concentration remained an approximately constant
5.73 g dry weight/L (D.W.) (Figure 2). In the course of
lipase synthesis R. delemar uses molasses sucrose as
both a carbon and energy source. Molasses sucrose
may be consumed to assimilate into biomass,
assimilate into an extracellular product, and as energy
for growth and maintenance, whereas sunflower oil is
used as an inducer and/or carbon source.
38
Biomass Concentration (g D. W./L)
The effects of the composition of growth medium and fermentation conditions on the production of lipase by R. delemar
8
0,5% (v/v)
1% (v/v)
2% (v/v)
4% (v/v)
6
4
2
0
0
2
4
6
Time (Day)
8
10
Figure 2. Time course of biomass concentration in fermentation
media containing various concentrations of sunflower
oil (pH = 8.0; temperature = 30 °C; concentration of
molasses sucrose = 1 g/L).
The effect of surface-active materials on lipase
activity
Surface-active materials either affect the
permeability of the cell, increasing the secretion of
lipase, or have a surfactant effect on cell-bound lipase.
To investigate the effect of surface-active materials on
lipase activity 2 different surface-active materials,
Tween-80 and rhamnolipid (biosurfactant), were used
as comparatives with tributyrin. Surface-active
materials were added to the fermentation media at
0.5% (v/v). As can be seen in Figure 1, higher enzyme
activity was observed in the fermentation medium
containing Tween-80, as compared to media
containing rhamnolipid or tributyrin. In the presence
of Tween-80 maximum lipase activity was 63.68 U/L,
which was approximately 13.5-fold greater than that
in the absence of the surface-active material.
Tween-80 seems to have a double effect on lipase
activity. Tween-80 can act as an inducer and is also a
surfactant. The presence of Tween-80 stimulates the
release of the enzyme, increasing lipase activity
recovered in the growth medium. Tween-80 can also
be used as a sole carbon source in the culture
medium, as it is miscible with water and does not
inhibit fungal growth. Lipase synthesis was reported
to be inhibited by high concentrations of oleic acid,
and thus by high concentrations of olive oil (30). In
the present study the lowest lipase activity (52.27 U/L)
was obtained when olive oil was added to the
Ü. AÇIKEL, M. ERŞAN, Y. SAĞ AÇIKEL
fermentation medium. Tween-80 provides oleic acid,
the intrinsic carbon source for cell growth and lipase
production via a mode of controlled release; therefore,
it decreases the repression of lipase synthesis. R.
delemar lipase activity in the presence of surfaceactive materials decreased in the order of Tween-80 >
tributyrin > rhamnolipid.
The other biological surface-active material used
in the present study was rhamnolipid (from the
glycolipids group) produced by Pseudomonas
aeruginosa. The effect of the commercial
biosurfactant rhamnolipid (supplied from Jeneil Co.)
as an inducer was not previously investigated. The
inclusion of rhamnolipid in the fermentation medium
resulted in lipase production 4.5-fold greater than that
observed in the control (no surfactant added).
Variation in the concentration of Tween-80 also
enhanced lipase production. Maximum lipase activity
(91.73 U/L) was observed when 1% (v/v) Tween-80
was added to the substrate as an additional carbon
source and inducer; however, lipase activity decreased
at higher Tween-80 concentrations. It was accepted
that the increase in fatty acid accumulation via
hydrolysis of the substrate suppressed lipase synthesis.
The inclusion of 0.5% and 1% (v/v) Tween-80 in the
fermentation medium resulted in an increase in
biomass production (2.96 and 3.93 g dry weight/L,
respectively), as compared to the control (0.55 g dry
weight/L) (no surfactant added). Increasing the
concentration of Tween-80 to 2% and 4 % (v/v)
decreased the biomass concentration to 2.18 and 2.13
g dry weight/L, respectively.
In order to overcome mass transfer limitations it
is very important to study the optimum stirring rate.
When stirring rates were increased from 50 to 250
rpm, lipase activity (103.12 U/L) increased as the
stirring rate increased from 50-200 rpm, and then
decreased as the rate increased to 250 rpm (Figure 3).
Enzymes are very sensitive to mechanical force. Shear
stress at higher stirring rates might disturb the
intricate shape of a complex molecule to such a degree
that enzyme denaturation occurs. The decrease in
enzyme activity observed at the highest stirring rate
tested in the present study may have been due to
perturbation of the protein structure during the
synthesis of lipase.
The presence of air is essential for lipase
production. The effect of oxygen limitation on lipase
production by pellet and filamentous cultures can be
significant. Oxygen can even be the limiting substrate
in shake flask cultures with pellet growth. Maximum
lipase activity (102.83 U/L) was obtained with a 2vvm aeration rate on the fourth day of fermentation.
At the 4-vvm aeration rate high lipase activity (97.4768.80 U/L) was maintained for a longer period of time
(between the 4th and 6th days of fermentation) in the
culture broth. The presence of aeration increased
lipase activity. A reduction in the fermentation time
due to an increase in the aeration rate was noted.
Moreover, enzyme activity occurred earlier and lasted
longer.
The effect of the stirring and aeration rates on
lipase activity
50 rpm
100 rpm
150 rpm
200 rpm
250 rpm
100
Activity (U/L)
Filamentous microorganisms such as molds often
form microbial pellets at high biomass concentrations
and in the absence of stirring the suspension culture.
Moreover, the morphology of R. delemar is normally
a pellet-like cake. Alternatively, filamentous cells can
grow on the surface of a moist solid or fermentation
liquid in the presence of insufficient stirring. The
transport of both substrate and inducer to the cells
inside pellets, and the release of enzyme produced
inside pellets to the growth medium may be subject
to diffusion limitations. This problem is the same that
occurs with bacteria or yeasts entrapped/immobilized
in spherical gel particles.
120
80
60
40
20
0
0
2
4
6
Time (Day)
8
10
12
Figure 3. Effect of stirring rate on lipase activity (pH = 8.0;
temperature = 30 °C; concentration of molasses sucrose
= 1 g/L; concentration of sunflower oil = 0.5% (v/v)).
39
The effects of the composition of growth medium and fermentation conditions on the production of lipase by R. delemar
The combined effect on enzyme activity of
molasses sucrose and sucrose as the carbon source
Although R. delemar utilizes a variety of carbon
and nitrogen sources for lipase production, molasses
was chosen in the present study due to its high sucrose
and other nutrient content, low cost, ready
availability, and ease of storage for industrial
production. As molasses is readily available as a waste
material of the sugar industry, enzyme production
costs can be reduced considerably when it is added to
fermentation media as a source of carbon, nitrogen,
and some other useful trace elements. To increase the
effect of the carbon source on enzyme activity, both
sucrose and molasses were added to fermentation
media. To prevent the inhibition effect of the
components of molasses other than sugar, such as
some undesired trace elements and metal oxides, the
molasses sucrose concentration should be kept at the
optimum value of 1.0 g/L. The sucrose concentration
was increased to 1-10 g/L in the fermentation media
containing 1.0 g/L of molasses sucrose. Maximum
lipase activity (92.27 U/L) was obtained in the
fermentation medium containing 5 g/L of sucrose on
the fourth day of fermentation (Figure 4). The
inclusion of 5 g/L of sucrose in the fermentation
medium resulted in lipase production that was 36.5fold greater than that obtained with the control (only
sucrose at the same concentration was added) and
lipase production 19.5-fold greater than that with the
fermentation medium to which 1.0 g/L of molasses
sucrose alone was added. The maximum biomass
concentration (7.95 g dry weight/L) was obtained in
the fermentation medium that contained 5.0 g/L of
sucrose (Figure 4). Sucrose concentrations higher
than 5.0 g/L inhibited both the enzyme activity and
biomass concentration.
The combined effect on enzyme activity of
molasses sucrose and sucrose as carbon sources,
sunflower oil as an inducer, and Tween-80 as a
surface-active material
Firstly, to observe the effect of sunflower oil alone
sunflower oil as an inducer was added to the
fermentation medium containing both molasses and
sucrose. Although maximum lipase activity was
observed on the 4th day of fermentation, relatively
high, stable, and long-term enzyme activity was also
observed between the 4th and 7th days of
fermentation. In the presence of 0.5% (v/v) sunflower
oil maximum lipolytic activity was 219.71 U/L, which
was approximately 86.8-fold greater than that in the
absence of molasses and sunflower oil, 46.4-fold
greater than that in the absence of sucrose and
sunflower oil, 2.36-fold greater than that in the
absence of sucrose, and 2.38-fold greater than that in
the absence of sunflower oil (Figure 5). The maximum
600
500
Activity (U/L)
80
7
6
60
5
1 g/L Activity
2 g/L Activity
5 g/L Activity
10 g/L Activity
1 g/L Biomass Conc.
2 g/L Biomass Conc.
5 g/L Biomass Conc.
10 g/L Biomass Conc.
40
20
0
2
4
6
Time (Day)
8
10
4
3
2
1
0
Figure 4. Effect of sucrose concentration on lipase activity and
biomass concentration in fermentation media
containing molasses sucrose and sucrose as main
carbon sources (pH = 8.0; temperature = 30 °C;
concentration of molasses sucrose = 1 g/L).
40
Activity (U/L)
8
0
Sunflower oil
Tween - 80+Sunflower oil
9
Biomass Concentration (g/L)
100
400
300
200
100
0
0
2
4
6
8
Time (Day)
10
12
Figure 5. Effects of sunflower oil and combined effects of
sunflower oil and Tween-80 on lipase activity in
fermentation media containing molasses sucrose, and
sucrose as main carbon sources (pH = 8.0; temperature
= 30 °C; concentration of molasses sucrose = 1 g/L;
concentration of sucrose = 5.0 g/L; concentration of
sunflower oil = 0.5% (v/v), concentration of Tween-80
= 1% (v/v)).
Ü. AÇIKEL, M. ERŞAN, Y. SAĞ AÇIKEL
biomass concentration was 7.74 g dry weight/L at the
end of the ninth day of fermentation, which was very
similar to the biomass concentration obtained in the
fermentation medium that contained both molasses
and sucrose (Figure 6).
Secondly, to stimulate the release of the enzyme,
Tween-80 was also added to the fermentation
medium containing molasses sucrose and sucrose as
carbon sources, and sunflower oil as an inducer. The
results show that lipase production was 2.30-fold
greater and lipase activity was 505.42 U/L when 1%
(v/v) Tween-80 was added to the medium (Figure 5);
however, mycelial growth decreased 1.48-fold (5.24 g
dry weight/L), as compared with the 7.74 g dry
weight/L in its absence (Figure 6).
The effect of activators on lipase activity
A range of different activators (NaCl, KCl, CaCl2,
MgCl2, gum arabic, and EDTA) was tested for their
ability to support growth and enzyme production by
R. delemar. For the purpose of observing the effect of
the activators on lipase activity, 0.5 g/L of activator
was added to the fermentation medium. Lipase
activity observed in the presence of activators
decreased in the order of NaCl > KCl > CaCl2 > MgCl2
> gum arabic > EDTA, and maximum lipase activity
(964.55 U/L) was obtained in the presence of NaCl. R.
The maximum biomass concentration (4.44 g dry
weight/L) was obtained in the presence of CaCl2 in
the fermentation medium. The biomass
concentrations obtained in the presence of activators
decreased in the order of CaCl2 > KCl > NaCl > gum
arabic > MgCl2 > EDTA.
The effect of heavy metal ions on lipase activity
To observe the effect of heavy metal ions on lipase
activity 0.1 g/L of metal ions were added to
+
+
2+
2+
fermentation media. Na , K , Ca , and Mg ions
enhanced R. delemar lipase activity, whereas Fe2+,
Mn2+, Co2+, and Ni2+ inhibited it. Lipase activity in the
presence of heavy metal ions decreased in the order
of Fe > Mn > Co > Ni (Figure 8). Salts of Co2+ and Ni2+
strongly inhibited lipase activity, suggesting that they
were able to alter enzyme conformation. Enzyme
activity in the media containing Fe2+ (85.23 U/L) and
8
1200
KCl
NaCl
CaCl2
MgCl2
Gum Arabic
EDTA
1000
6
Activity (U/L)
Biomass Concentration (g D. W./L)
10
delemar lipase activity increased 1.91-, 1.85-, 1.83-,
+
+
2+
2+
1.76-fold, respectively, when Na , K , Ca , and Mg
ions were added to the production medium (Figure
7). The enzyme required Na+, K+, Ca2+, and Mg2+ ions
as cofactors for catalytic activity. Lipase activity was
not affected by the metal chelator EDTA. As crude
enzyme samples taken from the fermentation
medium were used for the assay, EDTA might have
formed complexes with other compounds present in
the supernatant liquid phase, rather than with ions in
the lipase.
4
Sunflower oil
Tween-80+Sunflower oil
2
0
0
2
4
6
Time (Day)
8
10
Figure 6. Effects of sunflower oil and combined effects of
sunflower oil and Tween-80 on biomass concentration
in fermentation media containing molasses sucrose and
sucrose as main carbon sources (pH = 8.0; temperature
= 30 °C; concentration of molasses sucrose = 1 g/L;
concentration of sucrose = 5.0 g/L; concentration of
sunflower oil = 0.5% (v/v); concentration of Tween-80
= 1% (v/v)).
800
600
400
200
0
0
1
2
3
4
5
Time (Day)
6
7
8
Figure 7. Effects of activators on lipase activity (pH = 8.0;
temperature = 30 °C; concentration of molasses sucrose
= 1 g/L; concentration of sucrose = 5.0 g/L;
concentration of sunflower oil = 0.5% (v/v);
concentration of Tween-80 = 1% (v/v); concentration
of activator = 0.5 g/L).
41
The effects of the composition of growth medium and fermentation conditions on the production of lipase by R. delemar
100
Mn
Co
Fe
Ni
Activity (U/L)
80
60
40
20
0
0
2
4
Time (Day)
6
8
Figure 8. Effects of inhibitors on lipase activity (pH = 8.0;
temperature = 30 °C; concentration of molasses sucrose
= 1 g/L; concentration of sucrose = 5.0 g/L;
concentration of sunflower oil = 0.5% (v/v);
concentration of Tween-80 = 1% (v/v); concentration
of inhibitor = 0.1 g/L).
Mn2+ (71.25 U/L) was approximately equal to the
enzyme activity observed in the media containing
molasses as the sole carbon source, and sunflower oil
as the sole inducer or Tween-80 as the sole inducer.
Depending on the concentration of heavy metal ions,
enzyme activity, of course, increased or decreased.
Biomass concentrations in the presence of heavy
metal ions also decreased in the same order of Fe >
Mn > Co > Ni. The maximum biomass concentration
(5.51 g dry weight/L) occurred in the presence of Fe
ions; Ni ions strongly inhibited microorganism
growth and the biomass concentration decreased to
2.78 g dry weight/L.
Very little information is available on lipase
enzyme activity in the presence of heavy metals.
Reported results concerning the effect of heavy metals
on lipase activity are inconsistent. The following metal
ions are known inhibitors of lipase: Ag+, Al3+, Mn2+,
Sn2+, and Zn2+. Strong inhibitors are Hg2+ and Fe2+.
Ca2+ is commonly known as a lipase activator. Mg2+ is
also a known lipase activator, but Fe3+ and Cu2+ are
commonly known as inhibitors. All these metal ions,
as well as Ca2+, were reported to have an inhibitory
effect on the lipase activity of Flavobacterium
odoratum (31). Interestingly, it was also reported that
3 of these traditional inhibitory metal ions, Fe2+, Fe3+,
and Cu2+, as well as Mg2+ enhanced F. odoratum lipase
activity. The effects of metal salts on a thermostable
42
lipase from Pseudomonas sp. (KWI-56) were studied
(32). The enzyme was reported to be inhibited by
Cu2+, Hg2+, Sn2+, and Zn2+, but was not affected by
Ca2+. Lipase from Pseudomonas aeruginosa LP602 was
reported to be insensitive to many ions tested (i.e.
Cd2+and Co2+, the remaining activity of the enzyme
was higher than 80%), except Zn2+, which inhibited
activity by 48% (18). This lipase was assumed to be
stable against ions reported to inhibit other lipases (i.e.
Fe3+, Fe2+, and Hg2+). While the enzyme treated with
Ca2+ exhibited a slight increase in activity, the enzyme
remained insensitive to Na+, Ag+, and Ni2+ ions. The
enzyme also remained stable when treated with
EDTA. In contrast to our results, Pseudomonas
aeruginosa KKA-5 lipase activity was reported to
decrease by about 20%-30% in the presence of Na+
+
and K (group IA), whereas among the group IIA ions
(the alkaline-earth metals) Mg2+ and Ca2+ enhanced
it (33), providing results consistent with those
obtained in the present study.
Incubation of crude P. aeruginosa KKA-5 lipase in
metals like Mn2+ from group VIIB, and Fe2+, Ni2+, and
Co2+ from group VIIIB inhibited activity by 5%-15%,
similarly as in the present study. Fe2+, Fe3+, Hg2+, and
Cu2+ ions were reported to decrease Rhizopus oryzae
lipase activity (34). The presence of –SH groups in the
lipase molecule gives the enzyme intrinsic instability
and causes inactivation of the enzyme via oxidation
or interaction with certain heavy metal ions (35).
Heavy metal ions react with –SH groups in the side
groups of cysteine residues in the protein chain (36).
If cysteine residue is somewhere on the protein chain,
which affects the way it folds into its tertiary structure,
altering this group, it could change the shape of the
active site and the enzyme becomes deactivated.
Conclusion
The effects of different commercial oils and
tributyrin were observed on the production of R.
delemar lipase. In the presence of 0.5% (v/v)
sunflower oil maximum lipase activity was 93.07 U/L,
which was approximately 19.7-fold greater than that
observed in the basal medium. Amongst the various
surface-active agents tested in lipase production,
Tween-80 (1% (v/v)) was the best; an approximately
19.4-fold increase in lipase production was observed,
as compared to the basal medium.
Ü. AÇIKEL, M. ERŞAN, Y. SAĞ AÇIKEL
When the stirring rate was increased from 50 to
250 rpm lipase activity (103.12 U/L) increased up to
200 rpm, and then decreased. The effect of the
aeration rate was the same order of magnitude as that
of the stirring rate. Maximum lipase activity (102.83
U/L) occurred at the 2-vvm aeration rate.
To increase the effect of the carbon source on
enzyme activity the combined effect of molasses
sucrose and sucrose was investigated. Maximum
lipase activity of 92.27 U/L was observed at the 5-g/L
of sucrose concentration, which was approximately
36.5-fold greater than that observed in the absence of
molasses, and 19.5-fold greater than that observed in
the fermentation medium that did not contain
sucrose. Then sunflower oil (0.5% v/v) as an inducer
was added to the fermentation medium containing
both molasses and sucrose, and lipolytic activity
increased to 219.71 U/L. The presence of Tween-80
in the fermentation medium containing molasses
sucrose, sucrose, and sunflower oil stimulated the
release of the enzyme, increasing lipolytic activity
(505.42 U/L) in the culture medium 2.30-fold.
was observed in response to Ca2+ and Mg2+, whereas
2+
2+
Fe and Mn decreased enzyme activity. In the
presence of Fe ions lipase activity was reduced by 83%
(85.23 U/L). The most negative effect was noted in the
presence of Co2+ and Ni2+ salts. The enzyme appeared
to be insensitive to EDTA, whereas a 1.25-fold
increase in lipase activity occurred in the presence of
gum arabic. As a result, controlling the culturing
conditions and modifying the composition of the
medium dramatically improved the production of R.
delemar lipase.
Acknowledgements
The authors wish to thank TÜBİTAK (the
Scientific and Technological Research Council of
Turkey) for partial financial support of this study
(project no: MİSAG-282). We also wish to thank
Jeneil Biosurfactant Co. (Saukville, WI, USA) for
supplying the rhamnolipid biosurfactant.
Corresponding author:
The salts of Na+ and K+ had the most positive
effects on enzyme activity. In the presence of Na+ and
K+ lipase activity reached 964.55 and 934.41 U/L,
which was approximately 204-fold and 197-fold
greater, respectively, than that observed in the basal
medium that contained molasses sucrose only. Testing
bivalent cations showed that the greatest stimulation
Ünsal AÇIKEL
Department of Chemical Engineering,
Cumhuriyet University,
58140 Campus, Sivas - TURKEY
E-mail: uacikel@cumhuriyet.edu.tr
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