Optimisation of lipase and esterase assay for enzymatic extracts from subcutaneous adipose tissue of pig

Diana Martn*, Jorge Ruiz*, Mnica Flores**, Fidel Toldr**

*Tecnologa de los Alimentos, Facultad de Veterinaria, Universidad de Extremadura, Cceres, Spain **Instituto de Agroqumica y Tecnologa de los Alimentos (CSIC), Valencia, Spain

INTRODUCTION The assay of neutral lipase (NL), neutral esterase (NE) and acid esterase (AE) was optimised for porcine subcutaneous adipose tissue extracts. The optimised conditions for the assays of each enzymatic activity were applied in porcine subcutaneous adipose tissue extracts from pigs fed low (19%) or high (39%) levels of monounsaturated fatty acids (MUFA). MATERIAL AND METHODS 3g of sample were homogenized in 0.2 M Tris-HCl buffer (pH 8.2) containing 2 mg/mL sodium deoxycholate, 0.08 mg/mL Nonidet P-40, 0.05 mg/mL heparin, 10 mg/mL bovine serum albumine, 25 mM sucrose. After centrifugation (10000g 20min), the extracts were incubated in the reaction medium containing the fluorescent substrate 4-methylumbelliferyl oleate (lipases) or 4methylumbelliferyl propionate (esterases) at 37C for 20 min (lipases) or 10 min (esterases). The optimal quantity of enzymatic muscle extracts was determined by assaying different muscle extract dilutions at a constant substrate concentration. Subsequently, the Michaelis-Menten kinetic of each enzyme was elucidated by measurement of the activity at different substrate concentrations. The kinetic parameters, Vmax and Km, were calculated by the Lineweaver-Burk reciprocal plot. Fig.1. Enzymatic extract optimization
300 EE EE3/4 EE1/2 EE1/5 EE1/10

80 70 U( mol/L EE) 60 50 40 30 20 10 0 0

U( mol/L EE)

250 200 150 100 50 0 0

U( m ol/L EE)

EE EE3/4 EE1/2 EE1/5 EE1/10

350

Neutral lipase

300

EE EE3/4 EE1/2 EE1/5 EE1/10

Neutral esterase

Acid esterase

250 200 150 100 50 0

10

15

20

25

30 t(min)

10

15

20

25 t(min)

10

15

20

25 t(min)

Fig.2. Michaelis-Menten kinetic

3 V ( mol /L min) 2,5 2 1,5 1 0,5 0 0

Neutral lipase V ( mol /L min)

9 7,5 6 4,5 3 1,5 0 0

Neutral esterase V ( mol /L min)

9 7,5 6 4,5 3 1,5 0 0

Acid esterase

0,4

0,8

1,2 S (mM)

0,4

0,8

1,2 S (mM)

0,4

0,8

1,2 S (mM)

Table 1. Apparent Km and Vmax RESULTS AND DISCUSSION

Neutral lipase

Km (mM) 0.15 0.07 0.14

Vmax (mmol/L min) 2.82 9.23 12.84

The optimal quantity of enzymatic muscle extracts and Michaelis-Menten kinetic are shown in Fig.1 and 2, respectively. The optimal substrate concentration for each enzymatic activity was fixed as 5 times the obtained Km value (Table 1). These optimised conditions were successfully applied for the assays of lipase and esterase activities in porcine subcutaneous adipose tissue extracts from pigs fed low and high levels of MUFA (Fig. 3). This method revealed that, regardless the Moreover, lower activities of NL (p=0.003), NE (p=0.000) and AE (p=0.000) of subcutaneous adipose tissue from pigs fed high MUFA diets were observed respect to those from pigs fed low MUFA diets.
U(mol/L h)

Neutral esterase

Acid esterase

Fig. 3. Assay of lipases from pigs

24 20 16 12 8 4 0 NL NE AE High MUFA Low MUFA

MUFA level, the highest lipolytic activity was found for NE, followed by NL and AL.

Motilva M.; Toldr F.; Flores J. Assay of lipase and esterase activities in fresh pork meat and dry-cured ham. Z. Lebensm. Unters. Forsch. 1992, 195, 446-450.