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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., BrasilbCICECO, Departamento de Química, 3810-193, Universidade de Aveiro, Aveiro, Portugal

cDepartamento de Eng. Química, Universidade Federal do Ceará, 60455-760, CE, Brasil

INTRODUCTIONINTRODUCTION

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. 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.

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.

Cell Growth:

Cell growth was followed by optical density measurements at 570 nm converted to mg.mL -1 using a factor previously established.

Glucose:

Extracellular glucose concentration was determined by glucose oxidase method (Enzimatic Colorimetric Glucose Assay Kit, HUMAN GmbH - Germany.

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).

Protein analysis:

Protein was estimated by the Folin-Ciocalteau’s phenol reagent as outlined by Lowry et al. (1951), at 660 nm, using bovine serum albumin (BSA) as standard.

RESULTS AND DISCUSSIONRESULTS AND DISCUSSION

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.

RESULTS AND DISCUSSIONRESULTS AND DISCUSSION

3 4 5 6 7 8 9 100

20

40

60

80

100

120

Hyd

roly

tic

Act

ivit

y (p

NP

LU

/L)

pH of Buffers

Citrate buffer Na-phosphate buffer K-phosphate buffer Sodium bicarbonate buffer

Figure 1: Effect of pH reaction on activity (a) and stability (b) of Y. lipolytica lipase.

Effect of pH on activity and stability of lipase extract:

2 3 4 5 6 7 8 9 10 110

20

40

60

80

100

Resid

ual A

cti

vity

(%

)

pH

Contact time = 2 h Contact time = 24 h

25 30 35 40 45 50 55 60 65 700

10

20

30

40

50

60

70

80

90

100

Hyd

roly

tic

Act

ivit

y (p

NP

LU

/L)

Temperature of Reaction (ºC)0 50 100 150 200 250 300 350 400

0

20

40

60

80

100

120

Res

idu

al A

ctiv

ity

(%)

Time (hours)

Stability at 25ºC Stability at 37ºC Stability at 60ºC

0 500 1000 1500 2000 25000

10

20

30

40

50

Lipase B from Candida antarctica Lipase from Yarrowia lipolytica

Hyd

roly

tic

Act

ivit

y (p

NP

LU

/L)

Concentration of Substrate (M)

0

20

40

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140

MnCl2 1mM Glycerol 9% Ethanol 9% CaCl2 1mM EDTA 1mM Hexane 9% NaCl 1mM NaCl 100mM

Lipase B from Candida antarctica

Lipase from Yarrowia lipolytica

Figure 5: Effect of chemical compounds on activity of lipase extract produced by Y. lipolytica and type B from C. antarctica.

Figure 2: Effect of temperature on activity (a) and stability (b) of lipase from Y. lipolytica.

Effect of temperature on activity and stability of lipase extract:

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.

Effect of substrate concentration:

Effect of some additives:

Km = 0.234 mM

Km = 0.192 mM

0 50 200 2500

20

40

60

80

100

120

140

Res

idu

al A

ctiv

ity

(%)

Time (days)

Storage stability:

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.

CONCLUSIONSCONCLUSIONSThe 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.

MATERIALS AND METHODSMATERIALS AND METHODSStrain, 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:

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

28ºC

160 rpm

200 mL of YPD medium (w/v: Yeast extract 1%; Peptone 2%; Glucose 2%)