J. Basic Microbiol.

39 (1999) 1, 11 –15

(Departamento de Bioquímica, Universidade Estadual de Maringá, 87.020-900, Maringá, PR, Brazil)

Production of lipase by soil fungi and partial characterization

of lipase from a selected strain (Penicillium wortmanii)
MARIA APARECIDA FERREIRA COSTA and ROSANE MARINA PERALTA

(Received 6 July 1998/Accepted 9 September 1998)

Filamentous fungi from soil were screened for their ability to produce lipase. Among 56 filamentous
fungi tested, one strain identified as Penicillium wortmanii was selected as the highest lipase pro-
ducer. Maximum lipase production (12.5 U/ml) was obtained in 7-days cultures utilizing 5% (w/v)
olive oil as the carbon source. Optimum pH and temperature for crude lipase were 7.0 and 45 °C,
respectively. The enzyme was stable at 40 and 45 °C and it retained about 55% of its activity when
heated at 50 °C for 1 hour.

Lipases (triacylglycerol hydrolase, EC 3.1.1.3) are enzymes widely distributed among ani-
mals, plants and microorganisms that catalyse the hydrolysis of glycerol-ester bonds at fat
water interfaces. In anhydrous water-immiscible organic solvents they are also able to cata-
lyze the reverse reactions of synthesis and group exchange of esters and the resolution of
racemic mixtures into optically active alcohols or acids (TAIPA et al. 1992). These proper-
ties determine the biotechnological importance of lipases. A large number of lipases have
been screened for application as food additives (flavor-modifying enzymes), industrial reagents
(glyceride-hydrolysing enzymes) and cleaners (detergent additives) as well as for medical
applications (digestive drugs and diagnostic enzymes). Bacteria and especially fungi, are the
tools of choice for commercial production of lipases. The most important sources or com-
mercial lipase are Mucor, Rhizopus, Candida, Geotrichum, Aspergillus, Penicillium and
Humicola (GHOSH et al. 1996, GODTFREDSON 1990).
Most of the microbial lipases are extracellular, and are excreted through the cell mem-
brane into the culture medium. The amount of lipase produced depends upon several envi-
ronmental factors, such as temperature, pH, nitrogen, carbon and lipid sources, agitation and
dissolved oxygen concentration. Lipase production is generally stimulated by lipids and is
usually coordinated with the availability of triglycerides. Certain inducers also have a strong
effect on the stimulation of lipase production. The inducers include: triglycerides, free fatty
acids, bile salts and glycerol (GHOSH et al. 1996).
The purpose of this study was to investigate the ability to produce lipases of some fila-
mentous fungi isolated from soil and the effect of some environmental and nutritional fac-
tors on the production of lipases by a selected strain, Penicillium wortmanii. The extracel-
lular lipase of Penicillium wortmanii was studied regarding the optimum of both pH and
temperature, and its thermostability and pH stability.

Materials and methods

Screening for lipase-producing fungi: The fungi used were isolated from soil, during a screening
programme for lipase-producing microorganisms in this laboratory. Screening was carried out using
mineral medium (MONTENECOURT and EVEILEIGH 1977) with olive oil emulsion as the carbon source.
The fungi that showed a hydrolysis halo on this medium were selected for later lipase determina-
tion.
12 M. A. F. COSTA and R. M. PERALTA

Growth medium for lipase production: Enzyme production was studied in 250 ml ERLENMEYER
flasks containing 50 ml of mineral medium and different commercial oils (olive oil, sunflower oil,
and soybean oil), at 1 or 5% (w/v) as the carbon source. The cultures were incubated at 30 °C on a
rotary shaker at 120 rpm for up 10 days. The mycelia were removed from de media by filtration and
the lipase activity was analyzed in the culture filtrates.

Lipase assay: Lipase activity was determined by incubating a reaction mixture containing 5 ml of
olive oil emulsion (MACEDO et al. 1997), 2 ml of 0.1 M phosphate buffer, pH 7.0 and 1.0 ml of the
culture filtrate (lipase crude extract) at 37 °C for 30 min, with shaking of 130 rpm. After incubation,
the reaction was stopped by the addition of 15 ml of acetone-ethanol (1 : 1) and the liberated free fatty
acids were titrated with 0.05 N NaOH in the presence of phenolphtalein as indicator. One unit of
lipase activity was defined as the amount of enzyme which liberated 1 µmol of fatty acids per min.

Effect of pH on lipase activity and stability: The optimal pH was determined assaying the enzyme
at various pH on MCILVAINE’s buffers. pH stability was investigated by incubating the enzyme at
different pH for 1 h at room temperature. The remaining activities were measured under standard
conditions.

Effect of temperature on lipase activity and stability: The lipase assays were carried out at tempe-
ratures ranging from 25 to 70 °C. Thermal stability was investigated by incubating the enzyme at 30,
40, 45, 50 and 60 °C for 1 hour. Immediately afterwards the enzyme was immersed in an ice bath and
then the activity was tested under standard conditions.

Biomass determination: For biomass determination, the cultures broths were filtered, and solids
were washed twice with water and dried at 80 °C to a constant weight.

Results

A total of 56 filamentous fungi isolated from soil showed a halo of hydrolysis in basal me-
dium with olive oil emulsion as the only carbon source. Eight strains were found to produce
high activity of lipase in liquid media (Table 1). One strain, identified as Penicillium wort-
manii was selcted for further studies because it showed the highest productivity.
The influence of different commercial oils as carbon source at 1 and 5% on the produc-
tion of lipase by P. wortmanii was investigated (Table 2). Biomass production and lipase
activity were observed in all the cultures using oils as carbon source (soybean, olive, corn
and sunflower oil), and the maximum activity was reached with 5% olive oil as the carbon
source.

Table 1
Production of lipase by filamentous fungi from soil in liquid culture

Filamentous fungi Biomass (mg) Lipase activity (U/ml)

Aspergillus oryzae 507 ± 86 3.14 ± 0.25
Aspergillus niger isolated no. 1 470 ± 69 3.30 ± 0.28
Aspergillus niger isolated no. 2 540 ± 49 5.80 ± 0.60
Aspergillus fumigatus 320 ± 44 2.60 ± 0.21
Aspergillus flavus 580 ± 76 4.70 ± 0.62
Neurospora sp. 310 ± 24 3.50 ± 0.21
Rhizopus sp. 750 ± 83 5.70 ± 0.43
Penicillium wortmanii 550 ± 58 10.60 ± 0.92

The cultures were developed on 50 ml mineral medium using 5% olive oil as carbon source at 30 °C
on a rotary shaker at 120 rpm for 6 days. The results express the media ± SD of three different
experiments
Production of lipase by Penicillium wortmanii 13

Fig. 1
Production of lipase by Penicillium wort-
manii. The cultures were developed on
50 ml of mineral medium supplemented
with 5% olive oil as the carbon source
at 30 °C on a rotary shaker at 120 rpm
for up 10 days. Lipase activity (d); Bio-
mass (s)

Table 2
Production of lipase by P. wortmanii on different commercial oil

Commercial oil Biomass (mg) Lipase activity (U/ml)

1% corn oil 460 ± 34 6.30 ± 0.80

5% corn oil 610 ± 77 5.00 ± 0.60
1% olive oil 380 ± 27 5.10 ± 0.90
5% olive oil 540 ± 51 10.30 ± 1.80
1% sunflower oil 410 ± 39 1.30 ± 0.40
5% sunflower oil 620 ± 49 1.80 ± 0.20
1% soybean oil 430 ± 37 4.60 ± 0.50
5% soybean oil 670 ± 58 7.40 ± 0.60
1% glycerol 410 ± 50 0.35 ± 0.10
5% glycerol 720 ± 95 0.40 ± 0.10

The cultures were developed on 50 ml mineral medium using different commercial oil at 1 or 5% as
carbon source at 30 °C on a rotary shaker at 120 rpm for 6 days. The results express the media ± SD
of three different experiments

Fig. 2
Effect of pH on activity (d) and stability (s) of lipase
from P. wortmanii
14 M. A. F. COSTA and R. M. PERALTA

Fig. 3
Effect of temperature on activity (d) and
stability (s) of lipase from P. wortmanii

The microorganism was grown on 5% olive oil as the carbon source and the production
of lipase was determined as a function of time. As shown in Fig. 1, maximal lipase activity
(12.5 U/ml) was reached after 7 days of incubation, during the stationary growth phase. The
lipase production is related to biomass; when the biomass was in the maximum, lipase activity
was also high. Emulsification of culture media containing olive oil by gum arabic did not
increase the growth and lipase production by P. wortmanii (data not shown).
Some properties of crude lipase were studied. Crude lipase Penicillium wortmanii was
most active between pH 6.0 –8.0 and the enzyme was stable in pH range 5 to 10 at room
temperature after 1 hour of incubation at room temperature (Fig. 2).
The optimum temperature was 45 °C and it retained 98%, 90% and 55% of the initial
activity after 60 min at 40, 45 and 50 °C, respetively (Fig. 3).

Discussion
Many workers have exploited fungi as valuable sources of lipase due to the following pro-
perties: thermal stability, pH stability, substrate specificity and activity in organic solvents.
The use of fungi from soil to produce lipase is particularly interesting because heat resis-
tance has been demonstrated in different soil fungi (JESENKA et al. 1992, 1993, PIECKOVA
et al. 1994). In spite of lipases from different species of Penicillium have already been
purified and characterized (STOCKLEIN et al. 1993, ERDMANN et al. 1992, IBRIK et al. 1998,
PIMENTEL et al. 1994), this is the first report describing the production of lipase by a strain
of P. wortmanii isolated from soil.
The pH curve for P. wortmanii lipase was similar to other different microbial lipases
studied. Lipases are usually stable at neutral pH or near the neutral pH range of 6.0 to 7.5,
and with considerable stability at acidic pH down to 4.0 to alkaline pH up to 8.0 (GHOSH
et al. 1996, JESENKA et al. 1992, 1993, PIECKOVA et al. 1994).
Lipase from P. wortmanii showed high stability at 40 and 45 °C. Although the majority
of lipases from plants and animals lose completely the activity at temperature above 40 °C,
some microbial lipases have shown to be resistent to heat inactivation. The enzymes pro-
duced by Aspergillus niger, Rhizopus japonicus and Chromobacterium viscosum are stable
in solution at 50 °C and the thermotolerant Humicola lanuginosa excretes a lipase which is
stable at 60 °C (GHOSH et al. 1996). High thermostability have also been found in different
strains of Pseudomonas fluorescens (ANDERSON et al. 1979, KUMURA et al. 1993).
The characteristics of lipase from Penicillium wortmanii such as, stability of the enzyme
at 40 and 45 °C and a large range of pH, suggest its application in detergents and other
products that require a high stability at room temperature. Purification of lipase from
P. wortmanii is in progress in our laboratory.
Production of lipase by Penicillium wortmanii 15

Acknowledgements

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico
and Universidade Estadual de Maringá.

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Mailing address: Dr. ROSANE MARINA PERALTA, Departamento de Bioquímica, Universidade Esta-
dual de Maringá, 87.020-900, Maringá, PR, Brazil
E-mail: rmperalta@pbc.uem.br