EUROPEAN CONGRESS OF CHEMICAL ENGINEERING

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Characterization of an Extracellular lipase from
Yarrowia lipolytica
A.I.S. Brígidaa, P.F.F. Amarala, J.A.P.
Coutinhob, L.R.B. Gonçalvezc, M.A.Z. Coelhoa
aDepartamento de Eng. Bioquímica, Escola de
Química, Universidade Federal do Rio de Janeiro,
21949-900, R.J., Brasil bCICECO, Departamento de
Química, 3810-193, Universidade de Aveiro,
Aveiro, Portugal cDepartamento de Eng. Química,
Universidade Federal do Ceará, 60455-760, CE,
Brasil
INTRODUCTION
RESULTS AND DISCUSSION
Effect of pH on activity and stability of lipase
extract
Lipase (triacylglycerol eser hydrolases, E.C.
3.1.1.3) constitute a group of enzymes that
catalyse lipids hydrolysis as its biological
function.
Somes studies have reported the yeast Yarrowia
lipolytica, formerly known as Candida,
Endomycopsis or Saccharomycopsis lipolytica, as a
good lipase producer. This yeast is able to
produce extracellular, membrane-bound and
intracellular lipases encoded by approximating,
eighteen genes.
Therefore, the aim of this study was to
characterize the lipase activity of Yarrowia
lipolytica IMUFRJ 50682 (Baía de Guanabara
isolated strain, Brazil), produced under
submerged fermentation in a multiphase reactor,
due to its great importance for different
applications as catalysts in chemical processes.
Figure 1 Effect of pH reaction on activity (a)
and stability (b) of Y. lipolytica lipase.

• The lipase extract produced by Y. lipolytica
  IMUFRJ 50682 under different conditions
  (Pereira-Meirelles et al., 1997) showed a pH
  range of 6 10 and an optimum pH of 9.

Effect of temperature on activity and stability
of lipase extract
MATERIALS AND METHODS
Strain, Media and Culture Conditions Yarrowia
lipolytica IMUFRJ 50682 a wild type strain
isolated from Baía de Guanabara, Rio de Janeiro
Brazil, was kept at 4ºC on YPD-agar medium.
Pre-inoculum

• 28ºC
• 160 rpm
• 200 mL of YPD medium (w/v Yeast extract 1
  Peptone 2 Glucose 2)

EUROPEAN CONGRESS OF CHEMICAL ENGINEERING ECCE 6
Figure 2 Effect of temperature on activity (a)
and stability (b) of lipase from Y. lipolytica.

• Comparing LYL with a commercial enzyme
  (Lipozyme CALB L, Novozyme), both products have
  the same optimum temperature 37ºC.
• The studied extract revealed to be more stable
  then purified YlLip2 since it is capable to
  retain 86 of its initial activity for 5 h at
  37ºC while YlLip2 retained 83 for 4 h at 35ºC.

Production

• 28ºC
• 1 mg d.w. cells. mL-1
• 1,500 mL of YPD (w/v Yeast extract 1 Peptone
  0.64 Glucose 2)
• 20 (v/v) of perfluorodecalin

Effect of substrate concentration
Storage stability
Km 0.234 mM
Cell Growth Cell growth was followed by optical
density measurements at 570 nm converted to
mg.mL-1 using a factor previously established.
Km 0.192 mM
Glucose Extracellular glucose concentration was
determined by glucose oxidase method (Enzimatic
Colorimetric Glucose Assay Kit, HUMAN GmbH -
Germany.
Protein analysis Protein was estimated by the
Folin-Ciocalteaus phenol reagent as outlined by
Lowry et al. (1951), at 660 nm, using bovine
serum albumin (BSA) as standard.
Figure 3 Initial rate of pNPL hydrolysis by
lipase from Y. lipolytica and tye B from C.
antarctica.
Figure 4 Storage stability of crude extract of
lipase from Y. lipolytica at -10ºC.
Effect of some additives
Assay of hydrolytic activity with p-nitrophenyl
laurate (pNPL) The reaction occurred at 37 ºC
by the addition of 0.2 mL of enzyme solution to
1.8 mL of 560 µM pNP-laurate dissolved in 50mM
potassium-phospate buffer (pH 7.0), containing 1
(v/v) of dimethyl sulfoxide (DMSO).
Assay of hydrolytic activity with methyl
butirate Methyl butyrate hydrolysis was used to
determine the esterasic activity of LYL. The
reaction was initiated by the addition of 0.1 mL
of extract to 30 mL methyl butyrate solution
dissolved in 25 mM phosphate buffer pH 7.0.
Experiments were performed using an automatic
titrator (pHstat) and 50 mM NaOH as titrating
agent.
RESULTS AND DISCUSSION
Figure 5 Effect of chemical compounds on
activity of lipase extract produced by Y.
lipolytica and type B from C. antarctica.
Lipase Production The lipase extract produced
presebted specific activity of 22.4 pNPLU/g
protein, determined by p-nitrophenyl laurate
(pNPL) hydrolysis. However, for the hydrolysis of
methyl butirate, only 3 MBU/mL of extract was
found, which is a low activity value for this
method. By these results, it is posible to assume
that the amount of esterase in the extract is
minimum, or null. Therefore, the activity
expressed for pNPL hydrolysis is due to lipase
presence.
CONCLUSIONS
The crude lipase extract produced by Y.
lipolytica IMUFRJ 50682 showed to have similar
characteristics to other purified lipase extracts
from Y. lipolytica, mainly for YlLip2 strain
producer. Nevertheless LYL produced under
conditions reported in this paper possess
behaviour hardly similar to that obtained by the
same strain in different production conditions.