You are on page 1of 6

Biocatalysis and Agricultural Biotechnology ()

Contents lists available at ScienceDirect

Biocatalysis and Agricultural Biotechnology


journal homepage: www.elsevier.com/locate/bab

Original Research Paper

Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium


containing products and byproducts from the oleochemical industry
Georgi Dobrev a, Boriana Zhekova a,n, Valentina Dobreva b, Hristina Strinska a,
Pavlina Doykina a, Albert Krastanov c
a
Department of Biochemistry and Molecular Biology, University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria
b
Department of Engineering Ecology, University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria
c
Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd., 4002 Plovdiv, Bulgaria

art ic l e i nf o a b s t r a c t

Article history: The process of lipase biosynthesis by Aspergillus carbonarius in a submerged cultivation was studied. It
Received 1 July 2014 was established that inclusion of a lipid carbon source in the nutrient medium signicantly increased
Received in revised form enzyme production. Highest lipase activity was achieved when unrened rapeseed oil (195.61 U/L),
5 September 2014
soybean oil (155.00 U/L), and soap stock from sunower oil (149.00 U/L) were used as a carbon source.
Accepted 24 September 2014
These three raw materials were characterized by a high acid value, indicating high content of free fatty
acids. It was found that the high concentration of free fatty acids in the nutrient medium determined
Keywords: achievement of high lipase activity. Signicant increase in lipase biosynthesis was observed when
Lipase biosynthesis surfactants were included in the nutrient medium. In the presence of 20 g/L Tween 80 lipase activity was
Aspergillus carbonarius
more than 2-fold higher in comparison to a control sample. Lipase production was considerably
Oleochemical industry products
inuenced by the presence of organic nitrogen source in the medium. Meat extract was found to
Soap stock
Emulsifying agents stimulate enzyme biosynthesis.
& 2014 Elsevier Ltd. All rights reserved.

1. Introduction production by lamentous fungi varies according to the strain, the


composition of the growth medium and cultivation conditions
Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are class of (Cihangir and Sarikaya, 2004).
hydrolases which catalyze the hydrolysis of triglycerides to free fatty Microbial lipases are mostly extracellular and their production is
acids, diacylglycerols, monoacylglycerols and glycerol over an oil greatly inuenced by medium composition. Lipases are inducible
water interface (Reis et al., 2009). However, in the absence of water or enzymes and the major factor for the expression of enzyme activity
at micro-aqueous conditions, they catalyze the reverse reaction of was reported to be the carbon source. These enzymes are generally
synthesis of fatty acids esters (Kaushik et al., 2006). Lipases belong to produced in the presence of a lipid component such as oil or any
the structural super family of /-hydrolases whose activities rely other inducer, such as triacylglycerols, fatty acids, hydrolysable esters,
mainly on a catalytic triad usually formed by Ser, His and Asp residues. bile salts, and glycerol (Gupta et al., 2004; Sharma et al., 2001;
The serine residue usually appears embedded in the conserved Treichel et al., 2010). In the literature there are also results that
pentapeptide GlyXSerXGly (Fickers et al., 2011). inclusion of lipids in the nutrient medium does not stimulate lipase
Lipases are produced by plants, animals, and microorganisms. biosynthesis, and even it inhibits it (Adnan, 1998). For example
Microbial lipases have gained special industrial attention due to biosynthesis of lipase from Penicillium roqueforti was inhibited by
their stability, selectivity, and broad substrate specicity (Dutra butter, corn and olive oil (Eitenmiller et al., 1970).
et al., 2008; Griebeler et al., 2011). Many microorganisms are Some authors established a signicant stimulating effect of
known as potential producers of lipases, including bacteria, yeasts, various surfactants such as Tween 80 and lecithin on microbial
and fungi (Abada, 2008). lipase biosynthesis (Ghosh et al., 1996; Johri et al., 1990; Saxena
Some of the most commercially important lipase producing fungi et al., 1999). Surface-active materials either affect the permeability of
are recognized as belonging to the genera Rhizopus sp., Aspergillus sp., the cell, increasing enzyme secretion, or have a surfactant effect on
Penicillium sp., Geotrichum sp., Mucor sp., and Rhizomucor sp. Lipase cell-bound lipase. Tween-80 seemed to have a double effect on
lipase activity. It could act as an inducer and it was also a surfactant.
It can also be used as a sole carbon source in the culture medium, as
n
Corresponding author. Tel.: 359 32 603 605. it is miscible with water and does not inhibit fungal growth (Acikel
E-mail address: zhekova_b@yahoo.com (B. Zhekova). et al., 2011).

http://dx.doi.org/10.1016/j.bcab.2014.09.011
1878-8181/& 2014 Elsevier Ltd. All rights reserved.

Please cite this article as: Dobrev, G., et al., Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and
byproducts from the oleochemical industry. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.011i
2 G. Dobrev et al. / Biocatalysis and Agricultural Biotechnology ()

Lipase production by fungi strains is also signicantly inu- same conditions. For investigation of the effect of free fatty acids,
enced by the type and concentration of nitrogen source in the the composition of sunower oil was modied by addition of free
nutrient medium. According to some authors, C/N ratio has a great stearic acid in concentrations 3.33, 6.66, 10.00 and 13.33% (w/w). The
effect on enzyme biosynthesis (Lima et al., 2003). The use of modied sunower oil was used for lipase biosynthesis in concentra-
complex organic nitrogen sources such as peptone and yeast tion 15 g/l in the nutrient medium.
extract is widely practiced in submerged cultivation for microbial
lipase production (Adnan, 1998; Basheer, 2007; Fadiloglu and 2.5. Effect of surfactants on lipase biosynthesis
Erkmen, 2002; Rajan and Nair, 2011).
Over the recent years, research on the selection of suitable The effect of surfactants on lipase biosynthesis was investigated
substrates for biotechnological process has mainly been focused with two industrial preparations Tween 80 (Sigma) and Agrimul
on agroindustrial residues, due to their potential advantages. PG 2069 (Cognis Corporation) in the range of 5.030.0 g/L. Cultiva-
Utilization of agroindustrial wastes provides alternative substrates tion was performed in a medium with the following composition
and may help solving pollution problems, which otherwise might (g/L): unrened rapeseed oil 20.0, pepton 5.0, K2HPO4 2.5, KCl 0.5,
be caused by their disposal. On the other hand their use con- MgSO4 0.5 at the same conditions.
siderably reduces the cost of the produced enzyme preparations.
The choice of the substrate depends upon several factors, mainly 2.6. Effect of organic nitrogen source on lipase biosynthesis
related to cost and availability (Treichel et al., 2010).
The aim of the article is investigation on lipase biosynthesis in a The effect of organic nitrogen source on lipase biosynthesis was
submerged cultivation of Aspergillus carbonarius in a medium con- studied in a medium with the determined optimal composition (g/L):
taining products and byproducts from the oleochemical industry. unrened rapeseed oil 20.0, organic nitrogen source 5.0, K2HPO4 2.5,
KCl 0.5, MgSO4 0.5, Tween 80 (20.0 g/L). The inuence of meat
extract concentration was investigated in the range of 1.010.0 g/L at
2. Materials and methods the same conditions.

2.1. Microorganism
2.7. Determination of lipase activity

A strain of Aspergillus carbonarius NRRL369 from ARS Culture


For lipase activity determination the method proposed by
Collection was used for lipase biosynthesis. The strain was cultivated
Kaushik et al. (2006) was adapted. The method is based on the
on a slant agar medium containing (g/L): glucose 20.0, yeast extract
use of a synthetic substrate -nitrophenyl palmitate. Substrate
10.0, agaragar 15.0. The preparation of the medium included pH
solution was prepared by dissolving 30 mg -nitrophenyl palmitate
adjustment to 7.0 and sterilization at 121 1 for 30 min. The inocu-
in 10 mL isopropanol and addition of phosphate buffer with pH
lated tubes were incubated at 27 1C for 7 days and were stored at 4 1C.
6.0 to 100 mL total volume. 2.4 mL of the substrate solution were
incubated at 35 1 for 10 min and 0.1 mL suitably dissolved enzyme
2.2. Inocolum preparation was added. The enzyme reaction was performed at 35 1 for 30 min
and the enzyme was inactivated by addition of 1.0 mL 10% solution
For inoculum preparation the following nutrient medium was of NaOH. The reaction mixture was centrifuged at 3000 min 1 for
used (g/L): malt extract 10.0, yeast extract 4.0, glucose 4.0, K2HPO4 10 min and the absorbance at 405 nm was measured in accordance
1.0, NaNO3 2.5. Cultivation was performed in 500-ml Erlenmeyer to a reference sample with an inactivated enzyme.
ask, containing 100 mL nutrient medium with pH 6.5. The asks One unit (U) of lipase activity was dened as the amount of
were inoculated with 2.5 mL of 7 day-old culture, containing 3.107 enzyme releasing 1 mmol -nitrophenol for 1 min at 35 1 and
3.108 spores/mL and the strain was grown at 27 1C for 48 h with a pH 6.0.
180 rpm rotary shaking.
2.8. Analytical methods
2.3. Lipase biosynthesis
Determination of acid value, iodine value, peroxide value, and
Submerged cultivation of the strain was performed in 500-mL saponication value of vegetable oils was performed by using
Erlenmeyer asks, containing 50 mL nutrient medium. The com- standard methods (AOAC, 1997).
position of the medium varied according to the aim of the ex- Free fatty acids composition of soap stock was determined by gas
periment. After sterilization the medium was inoculated with chromatography with GC/FID Shimadzu chromatograph equipped
5.0 mL inoculum and the strain was grown at 27 1C for 64 h with with capillary column EC 30-Wax (30 m  0.25 mm  25 mm) and
a 180 rpm rotary shaking. Biomass was removed by ltration and ame ionization detector. The sample was transmethylated with 2 M
the culture liquid was tested for lipase activity. KOH in methanol (Christie, 2003). The analysis was performed at
130 1 initial temperature (4 min retention time), and gradient
2.4. Effect of carbon source on lipase biosynthesis temperature rise to 240 1 by 15 1/min (5 min retention time).
The temperature of the injector and the detector was 250 1. he
As carbon sources for lipase biosynthesis the following compo- mobile phase was hydrogen ow with rate 0.8 mL/min, split 50:1.
nents of the medium were tested: glucose, manose, starch, pectin, Biomass concentration was determined after drying at 110 1C to
carboxymethylcelulose (CMC), sunower oil, and combinations of a constant weigh.
sunower oil (10 g/L) and the respective carbohydrates (10 g/L).
The cultivation of the strain was performed in a medium with the
following composition (g/L): carbon source 10.0, peptone 5.0, K2HPO4 3. Results and discussion
2.5, KCl 0.5, MgSO4 0.5 at the above described conditions. When oils
were used as substrates, they were added as a sole carbon source. All 3.1. Effect of carbon source on lipase biosynthesis
oils were obtained by industrial manufacturers in Bulgaria. The
inuence of sunower oil, sunower soap stock and unrened The carbon source in the composition of nutrient media is one of
rapeseed oil was investigated in the range of 5.025.0 g/L at the the main factors affecting biosynthesis of extracellular microbial

Please cite this article as: Dobrev, G., et al., Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and
byproducts from the oleochemical industry. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.011i
G. Dobrev et al. / Biocatalysis and Agricultural Biotechnology () 3

enzymes. Most fungi strains require a lipid carbon source in the concentration of 20.0 g/L. Soap stock was characterized with higher
medium for lipase biosynthesis (Colla et al., 2010; Rajan and Nair, acid value 11.39 mg KOH/g in comparison to sunower oil 1.19 mg
2011; Rifaat et al., 2010). For these reasons different carbon sources KOH/g (Table 2). Probably the high content of free fatty acids in soap
separately and in a combination with a lipid substance were tested stock is the reason for the highest lipase activity. This consideration
for lipase biosynthesis from Aspergillus carbonarius (Table 1). was proved by experiments with modied sunower oil with
The results showed that inclusion of a lipid carbon source in addition of different concentrations of free stearic acid in the oil.
the composition of the culture medium is essential for lipase The results are presented in Fig. 2b. Increase of content of free fatty
biosynthesis. In all combinations with sunower oil lipase activity acids in oil to 6.66% led to enhancement in lipase activity. Higher
was detected. Similar results were reported for lipase biosynthesis concentrations of free fatty acids inhibited enzyme biosynthesis.
from Aspergillus sp. and Aspergillus niger NCIM584 maximum For further explanation of the results the fatty acid composition
enzyme activity was achieved in the presence of olive oil (Brooks of soap stock was determined (Table 3). Linoleic acid, oleic acid
and Asamudo, 2011; Hosseinpour et al., 2011). Lipase production and palmitic acid were the predominant fatty acids in its compo-
from Fusarium oxysporum was also stimulated by olive oil (Rifaat sition. It can be assumed that the inducing effect on lipase
et al., 2010). biosynthesis was due to the fatty acids with a chain length of
As Aspergillus carbonarius required a lipid source for lipase more than 16 carbon atoms. In the studied literature reports for
biosynthesis, the effect of different vegetable oils (sunower, soy- lipase biosynthesis from Aspergillus carbonarius by use of soap
bean, olive, unrened rapeseed, and corn) on enzyme production stock were rarely found. As soap stock is a waste product from the
was investigated (Fig. 1). In all further experiments they were used rening process of vegetable oils, the opportunity for its use for
as a sole carbon source. These oils are used as raw materials in the lipase biosynthesis has also environmental signicance.
food industry and are characterized by a signicantly high price. Due to the variability of soap stock composition, further experi-
Therefore a waste product from the production of rened sunower ments were carried out with unrened rapeseed oil as a carbon
oil (soap stock) was included in the study. Highest lipase activity was source. Rapeseed oil induced highest lipase production (Fig. 1). The
achieved in the presence of unrened rapeseed oil (195.61 U/L), effect of its concentration on lipase biosynthesis is presented in
soybean oil (155.00 U/L) and soap stock (149.00 U/L). Rapeseed oil Fig. 3. High lipase activity was obtained in the range of 1525 g/L
and soybean oil are obtained industrially in large quantities, and rapeseed oil, and maximum lipase biosynthesis (328.39 U/L) was
soap stock is a waste product, making them available for inclusion in achieved at 20.0 g/L.
the composition of the culture medium.
In order to nd a relationship between the quality parameters of
Sredi5200
the oils and lipase biosynthesis, the main characteristics of the oils
(acid value, iodine value, peroxide value, and saponication value)
Sunflower oil 41
Lipase activity (U/L)

were determined (Table 2). Comparing the results for lipase Oliv160oil 134.6355
biosynthesis (Fig. 1) with the characteristics of the oils (Table 2) it Corn120oil 80.55547
is seen that the three types of raw materials, giving highest lipase Soybean oil 155
activity (rapeseed oil, soybean oil, soap stock) were characterized by 80
Rapeseed oil 195.6107
high acid values. It can be concluded that the high content of free
Soap stock
40 149
fatty acids had strong inductive effect on lipase biosynthesis. This is
clearly seen in the study of the inuence of sunower oil and soap 0
stock concentrations on lipase biosynthesis (Fig. 2a). Soap stock was
found to have a much more pronounced inducing effect on lipase
biosynthesis compared with sunower oil. When using sunower
oil as a carbon source, a maximum lipase activity 54.87 U/L was
achieved at a concentration of 15 g/L. When soap stock was the
carbon source in the nutrient medium, maximum lipase activity
was about 4 times higher (232.74 U/L). It was achieved at a Fig. 1. Effect of vegetable oils on lipase biosynthesis.

Table 1
Effect of carbon source on lipase biosynthesis.

Carbon source Lipase activity (U/L) Biomass (g/L) Final Carbon source Lipase activity (U/L) Biomass (g/L) Final

Glucose 0 12.6 4.90 Sunower oil and glucose 36.0 12.8 3.56
Manose 0 11.7 4.80 Sunower oil and manose 32.5 12.1 3.85
Starch 0 13.5 6.30 Sunower oil and starch 31.5 13.5 3.65
Pectin 0 10.25 4.50 Sunower oil and pectin 32.3 12.2 3.72
CMC 0 10.8 6.20 Sunower oil and CMC 30.2 12.5 3.82
Sunower oil 38.0 12.4 3.60

Table 2
Quality parameters of raw materials.

Raw material Acid value (mg KOH/g) Iodine value (g I2/100 g) Peroxide value (meq/1000 g) Saponication value (mg KOH/g) Average Mw of fatty acids (g/mol)

Sunower oil 1.19 90.95 5.27 177.65 271.33


Olive oil 1.63 95.36 4.93 173.79 276.26
Corn oil 0.79 97.14 1.42 154.28 314.92
Soybean oil 9.70 101.41 4.45 148.67 329.79
Rapeseed oil 15.32 98.41 3.53 173.21 253.62
Soap stock 11.39 98.77 0.40 170.54 268.12

Please cite this article as: Dobrev, G., et al., Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and
byproducts from the oleochemical industry. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.011i
4 G. Dobrev et al. / Biocatalysis and Agricultural Biotechnology ()

800
250 700
Akt U/L
Sunflower oSoap stock Tween 80 Agrimul

Lipase activity (U/L)


600
200 5 36.88587 61 0.0 258.11
Lipase activity (U/L)

500
10 41.48267 84 5.0 434.55
150 400
15 54.86747 173.8197 10.0 575.16
300
100 20 41.61787 232.7432 20.0 677.91
200
25 38.56 102 30.0 656.95
100
50
0
0 0.0 5.0 10.0 15.0 20.0 25.0 30.0
0 5 10 15 20 25 30 Concentration (g/L)
Concentration (g/L)

Fig. 4. Effect of Tween 80 () and Agrimul PG 2069 () concentration on lipase


Lipase
250 activity U/L biosynthesis.
Stearic acid
0 52.36 0
Lipase activity (U/L)

200
0.05 115.65 3.33 was performed with 20.0 g/L rapeseed oil as a carbon source. It
150
0.1 218.65 6.66 should be noted that lipase activity in the presence of 20.0 g/L
100 0.15 97.35 10 Tween 80 was almost three times higher (677.91 U/L) compared to
0.2 86.35 13.33 the control without Tween 80 (258.11 U/L). Similar results were
50 obtained when Agrimul PG 2069 was used, maximal lipase activity
(704.27 U/L) was achieved in the presence of 30.0 g/L of the
0
0 3 6 9 12 15 surfactant.
Added stearic acid in sunflower oil (%) It should be noted that the stimulating effect of the surfactants
was achieved at high concentrations, which were comparable to the
Fig. 2. Effect of free fatty acids on lipase biosynthesis: (a) comparison between
carbon source concentration (20.0 g/L rapeseed oil). As their emul-
sunower oil () and soap stock (); (b) sunower oil with addition of sifying properties are displayed at much lower concentrations, it can
stearic acid. be assumed that they served also as nutrient substrates. However,
when Tween 80 was used as a sole carbon source in a concentration
Table 3
of 20 g/L, lipase activity was very low (6.00 U/L). This indicated that
Fatty acid composition of soap stock. Tween 80 did not act as an inductor on lipase biosynthesis, but most
likely the effect was due to the improved availability of the lipids in
Fatty acid Content (%) the medium.
Increase of lipase biosynthesis due to use of surfactants was
Butyric acid, 4:0 0.16
Caproic acid, 6:0 0.21 reported by other authors. Boekema et al. (2007) showed that
Caprylic acid, 8:0 0.23 inclusion in the medium of 0.1% Tween 80 increased lipase activity
Capric acid, C10:0 0.15 by 50%, in this case it functioned as an inducer of lipase operon of
Lauric acid, 12:0 0.10
the microbial strain. Acikel et al. (2011) reported that addition of
Tridecanoic acid, 13:0 0.11
Palmitic acid, 16:0 6.11
1% Tween 80 to the medium containing 0.5% sunower oil,
Palmitoleic acid, 16:1 0.09 increased lipase activity from 219.71 U/L to 505.42 U/L.
Stearic acid, 18:0 3.53
Oleic acid, 18:1 33.23
Linoleic acid, 18:2 56.08
3.3. Effect of nitrogen source on lipase biosynthesis

Most microbial lipase producing strains are saprophytes (Adnan,


Concentrat Akt, U/L 1998; Basheer, 2007), which determines that the type and concen-
350
5 45.9 tration of organic nitrogen source signicantly affect enzyme
300
10 65 biosynthesis. For these reasons the inuence of the organic nitrogen
Lipase activity (U/L)

250 15 301.62 source on lipase biosynthesis was studied (Fig. 5). The effect was
200 20 328.39 evaluated in comparison to a reference sample containing peptone
150 25 295 as nitrogen source.
100
Highest lipase activity was achieved when meat extract was
used as an organic nitrogen source. In this case enzyme activity
50
(1226.03 U/L) increased approximately 1.9 fold in comparison to
0 the reference (658.09 U/L).
0 5 10 15 20 25 30
No correlation between lipase activity and biomass concentration
Rapeseed oil (g/L)
was observed. When meat extract was used, Aspergillus carbonarius
formed high biomass content and produced high levels of lipase.
However, in the presence of peptone, soy peptone, gelatin peptone
Fig. 3. Effect of rapeseed oil concentration on lipase biosynthesis.
and casamino acids, biomass concentration was high, but lipase
biosynthesis was low.
3.2. Effect of surfactants on lipase biosynthesis The effect of meat extract concentration on lipase production is
presented in Fig. 6. Highest lipase activity was achieved at con-
The effect of emulsifying agents Tween 80 and Agrimul PG centrations of meat extract of 4.0 and 7.0 g/L. For further research,
2069 on lipase biosynthesis was studied (Fig. 4). The experiment the use of 4.0 g/L meat extract was chosen.

Please cite this article as: Dobrev, G., et al., Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and
byproducts from the oleochemical industry. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.011i
G. Dobrev et al. / Biocatalysis and Agricultural Biotechnology () 5

16
1200
14
1000

Lipase activity (U/L)


Akt, U/ml BM, g/l 12

Biomass (g/L)
Meat 800
extract 1226.027 14.2 10
Yeast extract 659.54 8.2 8
600
Malt extract 581.12 2.7 6
Pepton400 (Reference) 658.09 12.5
4
Soy pepton 318.83 15.2
200 2
Gelatin pepton 644.67 12.2
Casein 0hidrolysat 455.39 12.8 0
Trypton Meat Yeast Malt extract Pepton 5.4
362.1 Soy Gelatin Casein Trypton Casamino
extract extract (Reference) pepton pepton hidrolysat acids
Casamino acids 508.11 14.7
Lipase activity Biomass

Fig. 5. Effect of organic nitrogen source on lipase biosynthesis.

Acikel, U., Ersan, M., Acikel, Y., 2011. The effects of the composition of growth
1400 12 medium and fermentation conditions on the production of lipase by R. delemar.
1200 Turk. J. Biol. 35, 3544.
Lipase activity (U/L)

Meat extract, g/l Akt. U/l Biomas, g/l 10 Adnan, A., 1998. Fermentation pattern of fungal lipase and their application for
Biomass (g/L)

1000 esterication in nonaqueous media (Ph.D.). University of the Punjab, India.


1.0 707.97 6.9 8
800 Alford, J.A., Pierce, D.A., 1963. Production of lipase by Pseudomonas fragi in a
4.0 1227.14 12 6 synthetic medium. J. Bacteriol. 86, 2429.
600
7.0 1229.84 11.3 4 AOAC, 1997. Association of Ofcial Analytical Chemists. Ofcial methods of analysis.
400 17th ed. Washington, DC.
10.0 464.61 7.3 2 Basheer, S., 2007. Lipase production by marine fungus Aspergillus awamori (Ph.D.).
200
Cochin University of Science and Technology, India.
0 0 Boekema, B., Beselin, A., Breuer, A., Jaeger, K., Tommassen, J., 2007. Hexadecane and
1.0 4.0 7.0 10.0 Tween 80 stimulate lipase production in Burkholderia glumae by different
Meat extract (g/L) mechanisms. Appl. Environ. Microbiol. 73, 38383844.
Brooks, A., Asamudo, N., 2011. Lipase production by strains of Aspergillus sp.
Lipase activity Biomass isolated from contaminated body creams. J. Toxicol. Environ. Health. Sci. 3,
311316.
Christie, W., 2003. Lipid Analysis, third ed. The Oily Press, Bridgwater.
Cihangir, N., Sarikaya, E., 2004. Investigation of lipase production by a new isolated
Fig. 6. Effect of meat extract concentration on lipase biosynthesis. of Aspergillus sp. World J. Microbiol. Biotechnol. 20, 193197.
Colla, L., Rizzardi, J., Pinto, M., Reinehr, C., Bertolin, T., Costa, J., 2010. Simultaneous
production of lipases and biosurfactants by submerged and solid-state biopro-
cesses. Bioresour. Technol. 101, 83088314.
Similar results for stimulation of lipase biosynthesis by organic Dutra, J.C.V., Terzi, S.C., Bevilaqua, J.V., Damaso, M.C.T., Couri, S., Langone, M.A.P.,
nitrogen sources were reported for other microbial strains (Alford Senna, L.F., 2008. Lipase production in solid state fermentation monitoring
biomass growth of Aspergillus niger using digital image processing. Appl.
and Pierce, 1963; Freir et al., 1997; Hosseinpour et al., 2011). Biochem. Biotechnol. 147, 6375.
Aspergillus carbonarius strain is a promising producer of micro- Eitenmiller, R.R., Vakil, J.R., Shahani, K.M., 1970. Production and properties of
bial lipase. Enzyme biosynthesis was induced by inclusion of a Penicillium roqueforti lipase. J. Food Sci. 35, 130133.
Fadiloglu, S., Erkmen, O., 2002. Effects of carbon and nitrogen sources on lipase
lipid carbon source in the nutrient medium and highest enzyme production by Candida rugosa. Turkish J. Eng. Env. Sci. 26, 249254.
activity was achieved when lipid inductor was characterized by Fickers, P., Marty, A., Nicaud, J.M., 2011. The lipases from Yarrowia lipolytica:
high content of free fatty acids. Suitable substrates for lipase genetics, production, regulation, biochemical characterization and biotechno-
logical applications. Biotechnol. Adv. 29, 632644.
production were unrened rapeseed oil, soybean oil, and sun-
Freir, D.M., Teles, E.M.F., Bon, E.P.S., Sant Anna Jr, G.L., 1997. Lipase production by
ower soap stock. Surfactants signicantly inuenced enzyme P. restrictum in bench scale fermentation. Appl. Biochem. Biotechnol. 63-65,
biosynthesis and in the presence of Tween 80 enzyme activity 409421.
increased two folds. Tween 80 did not act as an inducer of lipase Ghosh, P., Saxena, R., Gupta, R., Yadav, R., Davidson, S., 1996. Microbial lipases:
production and application. Sci. Prog. 79, 119157.
biosynthesis, but the positive effect was probably due to the Griebeler, N., Polloni, A.E., Remonatto, D., Arbter, F., Vardanega, R., Cechet, J.L., di
formation of ne emulsion, improving the assimilation of a lipid Luccio, M., de Oliveira, D., Treichel, H., Cansian, R.L., et al., 2011. Isolation and
or increasing membrane permeability. Lipase production was screening of lipase producing fungi with hydrolytic activity. Food Bioprocess
Technol. 4, 578586.
considerably affected by the presence of organic nitrogen source Gupta, R., Gupta, N., Rathi, P., 2004. Bacterial lipases: an overview of production,
in the medium. Meat extract was found to stimulate enzyme purication and biochemical properties. Appl. Microbiol. Biotechnol. 64,
biosynthesis. 763781.
Hosseinpour, M., Najafpour, G., Younesi, H., Khorrami, M., 2011. Submerged culture
studies for lipase production by Aspergillus niger NCIM 584 on soya our.
Midle-East J. Sci. Res. 7, 362366.
Johri, B.N., Alurralde, J.D., Klein, J., 1990. Lipase production by free and immobilized
Acknowledgment
protoplasts of Sporotrichum-thermophile apinis. Appl. Microbiol. Biotechnol. 33,
367371.
This work was nancially supported by Bulgarian Science Fund, Kaushik, R., Saran, S., Isar, J., Saxena, R., 2006. Statistical optimization of medium
components and growth conditions by response surface methodology to
Ministry of Education and Science (Project Young Scientist 03/59
enhance lipase production by Aspergillus carneus. J. Mol. Catal. B: Enzym. 40,
2011). 121126.
Lima, V., Krieger, N., Sarquis, M., Mitchell, D., Ramos, L., Fontana, J., 2003. Effect of
nitrogen and carbon sources on lipase production by Penicillium aurantiogri-
References seum. Food Technol. Biotechnol. 41, 105110.
Rajan, A., Nair, A., 2011. A comparative study on alkaline lipase production by a
newly isolated Aspergillus fumigatus MTCC 9657 in submerged and solid-state
Abada, E.E., 2008. Production and characterization of a mesophilic lipase isolated fermentation using economically and industrially feasible substrate. Turk.
from Bacillus stearothermophilus AB-1. Pak. J. Biol. Sci. 11, 11001106. J. Biol. 35, 569574.

Please cite this article as: Dobrev, G., et al., Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and
byproducts from the oleochemical industry. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.011i
6 G. Dobrev et al. / Biocatalysis and Agricultural Biotechnology ()

Reis, P., Holmberg, K., Watzke, H., Leser, M.E., Miller, R., 2009. Lipases at interfaces: Sharma, R., Chisti, Y., Banerjee, U.C., 2001. Production, purication, characterization,
a review. Adv. Colloid Interface Sci. 147148, 237250. and applications of lipases. Biotechnol. Adv. 19, 627662.
Rifaat, H., El-Mahalawy, A., El-Menofy, H., Donia, S., 2010. Production, optimization Treichel, H., de Oliveira, D., Mazutti, M.A., di Luccio, M., Oliveira, J.V., 2010. A review
and partial purication of lipase from Fusarium oxysporum. J. Appl. Sci. on microbial lipases production. Food Bioprocess Technol. 3, 182196.
Environm. Sanit. 5, 3953.
Saxena, R., Ghosh, P., Gupta, R., Davidson, W., Bradoo, S., Gulati, R., 1999. Microbial
lipases: potential biocatalysts for the future industry. Curr. Sci. 77, 101115.

Please cite this article as: Dobrev, G., et al., Lipase biosynthesis by Aspergillus carbonarius in a nutrient medium containing products and
byproducts from the oleochemical industry. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.09.011i