Enzyme and Microbial Technology 38 (2006) 756–759

Production and down-stream processing of an extracellular

lipase from the yeast Yarrowia lipolytica
P. Fickers a,∗ , M. Ongena a , J. Destain b , F. Weekers c , P. Thonart a,b
aCentre Wallon de Biologie Industrielle, Service de Technologie Microbienne, Université de Liège,
Bd du Rectorat Bat. 40, B-4000 Liège, Belgium
b Centre Wallon de Biologie Industrielle, Unité de Bio-Industries, Faculté Universitaire des Sciences Agronomiques de Gembloux,
Passage des Déportés, 2, B-5030 Gembloux, Belgium
c Artechno S.A., Rue François Bovesse, 1, B-5030 Gembloux, Belgium

Received 5 January 2005; received in revised form 20 July 2005; accepted 2 August 2005

Abstract

Lipase constitutes an interesting class of enzyme with many biotechnological applications. However, the development of a fruitful process
must be set up to obtained a product compatible with the industrial and commercial needs. Here, we report the development of such a process
for the extracellular lipase secreted by the yeast Yarrowia lipolytica. The enzyme production, carried out in a 2000 L bioreactor, led to a lipase
activity of approximately 1100 U mL−1 after 53 h of fermentation. The post-culture treatment, consisting of a centrifugation, a filtration and
an ultra-filtration steps, led to 15-fold volume reduction and a 8-fold increased of the lipase activity. Finally, addition of 12% of milk powder
and 3% of gum arabic before spray-drying dehydration led to a stable powder with a lipolytic activity of 37,500 U g D.W.−1 .
© 2005 Elsevier Inc. All rights reserved.

Keywords: Lipase; Bioreactor; Down-stream process; Yarrowia lipolytica

1. Introduction addition of oleic acid compared to the wild-type strain and

Lipases (EC 3.1.1.3.) constitute a group of enzymes
having the ability to hydrolyze triglycerides at lipid–water
interfaces [1]. Interest in lipases has been greatly increased
during recent years, mainly because this class of enzymes
presents a broad range of biotechnological applications
[2–4]. However, few of them reported the development of
a fruitful process for extracellular lipase production. The
yeast Yarrowia lipolytica produce an extracellular lipase
encoded by LIP2 gene (Accession no. AJ012632). The 334-
amino-acid precursor protein is processed by the KEX2-like
endoprotease encoded by the XPR6 gene. The secreted
lipase is a 301-amino-acid glycosylated polypeptide with an
optimal catalytic activity at 37 ◦ C and pH 7 [5,6]. A chemical
mutagenesis on Y. lipolytica CBS6303 led to the isolation of
the LgX64.81 mutants selected on the basis of its phenotypic
characteristics, i.e. high level of lipase production upon
a LIP2 expression uncoupled from the glucose catabolite
repression [7,8]. Upon growth of LgX64.81 mutant in a
15 L bioreactor, lipase activity reached 1000 U mL−1 , which
represents a 35-fold increase compared to the parental
strain (Fickers et al., unpublished results). However, if these
enzymes are to become successfully commercialized in
the future, large-scale fermentation process must be set up
together with the development of an adequate down-stream
process ensuring an acceptable level of enzyme stability.
In this paper, the development of a lipase production pro-
cess in large-scale bioreactor is described. The down-stream
process was also investigated with the aim to obtain a stable
lipase powder with high enzymatic activity.
2. Materials and methods
2.1. Organism and growth conditions

∗ Corresponding author. Tel.: +32 4 366 28 61; fax: +32 4 366 28 62. The Y. lipolytica strain used in this study is the lipase
E-mail address: p.thonart@ulg.ac.be (P. Fickers). over-producing mutant LgX64.81 [5]. Pre-cultures were car-

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.08.005
 P. Fickers et al. / Enzyme and Microbial Technology 38 (2006) 756–759
ried out successively in 500 mL, 4 and 15 L of YPD medium
(glucose, 1.5%; yeast extract, 1%; casein peptone, 1%) at
29 ◦ C for 16 h. The latter pre-culture was performed in a 20
L bioreactor (LSL Biolafitte, Monze-Sur-le-Mignon, France)
at 29 ◦ C with a stirring speed of 350 rpm and an air flow
of 0.5 vvm. Lipase production were conducted in a 2000 L
LSL Biolafitte bioreactor filled with 1100 L of an optimized
medium containing: glucose, 1.5%; whey powder, 3%; yeast
extract, 3%; corn steep liquor, 1% (v/v); olive oil, 0.5%;
(NH4 )2 SO4 , 0.8% [5]. Fermentations were carried out at
29 ◦ C with a stirring speed of 120 rpm and an air flow of
0.7 vvm. Dissolved oxygen was continuously monitored with
an oxygen probe (Ingold, Urdof, Switzerland) and a level
probe, placed 30 cm from the top of the vessel, activated the
addition of antifoam (Tego KS911, Goldschmidt, Essen, Ger-
many) when necessary; pH was automatically maintained at
6.5 ± 0.1 by addition of 4N NaOH or 4N H3 PO4 .
2.2. Analytical procedures
Biomass were estimated by counting the cells on a Bürker
microscope slide on samples extracted with two-fifths of
propanol/butanol mixture (1:1, v/v) in case of culture in olive
oil containing media. The dry weight of either liquid and
solid lipase mixtures were performed on a infra-red mois-
ture balance. Lipase activity was measured by a titrimetric
method as previously described [5]. One unit of lipase is the
amount of enzyme that catalyzes the release of 1 ␮mol of
fatty acid per min at 37 ◦ C. Glucose concentration in the cul-
ture supernatant was determined using the Biomérieux kit
PAP7500 (Biomérieux, Marcy L’étoile, France) according to
the manufacturer’s recommendations.
2.3. Enzyme characterization
For pH stability determination, 1 g of spray-dried lipase
(1000 U/g protein) or 1 mL (1000 U mL−1 ) of supernatant
were dissolved in 100 mL of 100 mM sodium acetate buffer
pH 3 or in Na/K phosphate buffer pH 4–8. Residual lipase
activities were determined at 37 ◦ C as described above after
1 h of incubation. For thermo-stability determination, lipase
samples were diluted in 100 mM phosphate buffer pH 7 and
incubated at 10, 20, 30, 37, 45, 50 and 55 ◦ C for 1 h. After
cooling on ice, the residual lipase activity were determined
as described above. The protein concentration was estimated
using the Bio-Rad (Ivry-Sur-Seine, France) protein assay
reagent with bovine serum albumin as standard. Relative
enzyme activities are means of two experiments.
2.4. Down-stream fermentation steps
Cells were removed from the culture broth on a BTPX205
continuous centrifuge (Alfa Laval, Sweden) at 13,000 × g
at a flow rate of 500 L h−1 . The supernatant was then clari-
fied through a plate filter with 50 plates (SA997, Filtreclair,
France) of 0.13 m2 each, 3.5 mm thickness and 0.2 ␮m poros-
757
ity before being concentrated on a Niro UF/MF ultra-filtration
apparatus equipped with a 10 m2 polysulfone membrane (cut-
off of 10 kDa, Kolding Gruppen, Denmark). The dehydration
of the concentrated culture supernatant was performed on a
Niro P6.3 industrial spray-drier (Denmark) with inlet and out-
let temperature of 150 and 75 ◦ C [9], respectively, at a flow
rate of 20 L h−1 .
3. Results
3.1. Lipase production
During lipase production, cell growth and enzymatic activ-
ity were measured at various time points over 70 h (Fig. 1).
The biomass increased during the first 30 h of culture to
reach a concentration of 1.3 × 109 cells mL−1 . After 70 h
of fermentation, the increase of the dissolved oxygen value
together with the glucose exhaustion in the culture broth
indicated the end of the culture (Fig. 1). The production
of lipase exhibited two distinct phases. A high production
level of 34 U mL−1 h−1 was observed during the growth
phase, between 6 and 38 h of culture, while a production rate
of 5 U mL−1 h−1 was observed during the stationary phase
(Fig. 1). The maximal lipase activity of 1118 U mL−1 was
obtained after 53 h of culture. This lipolytic activity remained
constant until the end of the culture (70 h).
3.2. Post-culture treatment
After 70 h of fermentation, the culture broth was sub-
mitted to several down-stream steps. For each of them, dry
weight content and lipase activity were determined (Table 1).
A volume of 950 L of supernatant with a lipase activity of
12,692 U g D.W.−1 was recovered by continuous centrifu-
Fig. 1. Cell growth and lipase production by Y. lipolytica LgX64.81 during
culture in 2000 L bioreactor. Cell growth (
) is expressed in 107 cells mL−1
and lipolytic activity (䊉) in U mL−1 . Dissolved oxygen () and glucose
concentration () were multiplied by a factor of 20 and 100, respectively.
758 P. Fickers et al. / Enzyme and Microbial Technology 38 (2006) 756–759

Table 1 Table 3
Evolution of the lipase activity during the down-stream process Effect of additives on the lipase activity before and after spray-drying
Down-stream step Volume (1) D.W. (%) Lipase activity Additives Before After Yield (%)
U g D.W.−1
6% MP 12898 9862 76
Culture broth 1100 – ND 12% MP 8687 7522 86
Centrifugation 950 7.6 12828 12% MP + 3% GA 7857 6667 84
Plate filtration 950 5.6 13160 12% MP + 6% GA 6751 5572 82
Ultra-filtration 75 9.0 108166
Lipase activities, expressed in U mL−1 , are means of two experiments. MP,
Yields in lipase activity were calculated based on the activity expressed in milk powder; GA, gum arabic.
U g D.W.−1 . D.W., dry weight.
LgX64.81 mutant since addition of 2% of CaCl2 led to a
gation before being clarified by filtration through 0.2 ␮M four-fold increase in the lipase activity (Table 2).
porosity filtration plates. This latter, allowing the elimina- For the lipase dehydration, milk powder (MP), a source
tion of cell fragments and insoluble compounds (i.e. olive of both casein and calcium ions and gum arabic were added
oil and antifoam), led to the reduction by a one-third of the to the concentrated supernatant. Lipase activity was mea-
dry matter content of the solution without any loss of the sured before and after spray-drying (Table 3). Addition of
enzymatic activity (Table 1). The clarified culture supernatant 12% of milk powder led to highest lipase recovery, while
was then concentrated by ultra-filtration to a volume of 75 L addition of both 12% of milk powder and 3% gum arabic
with an eight-fold increase in lipase activity, indicating that led to a fluent powder more compatible with a commercial
almost all the enzyme activity was conserved. By contrast, product. The dehydration of the concentrated supernatant in
the small increase of the dry matter content of the solu- those conditions yielded to a powder with a lipolytic activity
tion after ultra-filtration show that small molecules, such as of 37,500 U g D.W.−1 .
medium compounds or cell metabolites are not retained by the
membrane. 3.4. Optimal activity and stability of the spray-dried
lipase
3.3. Effect of additives and lipase dehydration
The catalytic properties of the dehydrated lipase was
To minimize enzyme loss during dehydration, different investigated by comparing the enzymatic activity in the enzy-
stabilizing additives compatible with industrial needs were
tested to increase the thermal stability and/or the enzymatic
activity of the lipase. Carbohydrates, such as glucose, mal-
todextrine or starch, commonly used in spray-drying pro-
cesses, had no effect on the lipase activity (data not shown).
By contrast, addition of proteins, such as albumin or casein
presented a positive effect on the enzymatic activity of the
formulated lipase. An increase in the enzymatic activities of
1.63- and 2-fold was obtained in the presence of 1% of bovine
albumin and casein, respectively (Table 2). These molecules,
which can hydrogen bond to the lipase, could be facilitated
the interaction with hydrophobic substrates at the lipid–water
interface.
From crystallographic data, it was shown that many lipases
have a Ca2+ binding motif around the catalytic site (for
review, see [10]) and the presence of calcium was shown
to increase the thermo-stability or the catalytic activity of
some bacterial lipases [11,12]. This positive effect was
also observed for the extracellular lipase from Y. lipolytica

Table 2
Effect of additives on extracellular lipase activity
Concentration (%) Albumin Casein Concentration (%) CaCl2
0 100 100 0 100
0.5 146 203 2 408
1.0 163 201 5 384
Fig. 2. Temperature and pH stability of Y. lipolytica extracellular lipase after
Relative enzyme activities expressed in U mL−1 are means of two experi- dehydration by spray drying (grey) and in the culture supernatant (white).
ments. Relative enzyme activities are means of two experiments.
 P. Fickers et al. / Enzyme and Microbial Technology 38 (2006) 756–759
matic powder and in the concentrated supernatant. Samples
were incubated at different temperatures and different pH and
the residual lipolytic activities were determined. No signifi-
cant difference could be observed in the optimal catalytic tem-
perature between the spray-dried lipase and non-dehydrated
lipase (Fig. 2). The optimal activity was found at 37 ◦ C con-
firming previous observations [5]. By contrast, significant
differences were observed in the pH stability of the enzyme.
Both the spray-dried and the non-dehydrated lipase presented
an optimal catalytic activity at pH 7 but surprisingly the
spray-dried lipase seemed less sensitive to acidic pH com-
pared to the culture supernatant. This increased stability of
the spray-dried lipase in acidic conditions could be related to
the stabilization and neutralization of the lipase enzyme by
the casein peptide.
For the stability determination, lipase powders from four
independent spray-drying experiments were vacuum packed
and stored at 4 ◦ C. Periodic enzyme assays revealed a high
stability of the powder in time as a 2.7% loss of activity
was measured after 12 months of conservation under these
conditions (data not shown).
4. Discussion
The aim of the first part of this work was to set up a large-
scale lipase production process compatible with the industrial
needs from a non-GMO Y. lipolytica over-producing strain.
The yield in lipase production was found in accordance with
the 1175 and 1200 U mL−1 obtained previously in optimized
15 and 500 L bioreactor cultures, respectively ([5]; Fickers et
al., unpublished results). This demonstrates that an adequate
scaling-up of the lipase production was achieved and that
the culture parameters (aeration, stirring) are optimal in our
conditions. The yield of extracellular lipase production by
the LgX64.81 strain is more than 37-fold higher than those
observed previously for other Y. lipolytica isolates [13,14].
The ultra-filtration step allowed a significant reduction of the
working volume without any loss of enzyme activity. This is a
major advantage in the development of an industrial process.
In addition, this ultra-filtration contributed to a partial lipase
purification.
A common impediment to the production of commercial
enzyme is their low stability in aqueous solutions. Conse-
quently, drying is one possible way for the obtainment of
stable and storable enzymes. Spray-drying has been used
for the dehydration of many enzymes, such as proteases
or cellulases and offers cost advantages compared to
freeze-drying. However, dehydration by spray-drying is
a stress to proteins, causing their unfolding by thermal
denaturation and leading to the loss of enzymatic activity.
This unfolding is usually minimized by using additives,
such as carbohydrate polymers. Rather than using these
carbohydrates, milk powder and gum arabic were found
more suitable for the formulation of the Y. lipolytica lipase. In
addition to its low cost, milk powder presents the advantage
759
to be a source of both casein and calcium ions. The positive
effect of the Ca2+ on the lipase from Y. lipolytica is in
accordance with previous reports on the lipase from Candida
paralipolytica [15]. It was suggested that the role of calcium
ions was to remove fatty acid formed during the hydrolysis
of triglycerids as insoluble calcium soaps. In addition, the
recovery of enzymatic activity after dehydration and its
conservation during time were higher that those observed
for other hydrolase with maltodextrine, polyethyleneglycol
(PEG6000) or lactose as additives [16,17].
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