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 Lipase Activity as a Function of Interfacial
Tension Using the Rising Drop Method
on a New Oil Drop Tensiometer.
Claus Ladefoged*, Alain Cagna§ & Erik Gormsen*.
*Detergent Enzyme Division, Novo Nordisk , 2880 Bagsvaerd, Denmark.
§IT Concept, Parc de Chancolan, 69770 Longessaigne, France.

Summary

Using a recently developed computer controlled oil drop tensiometer it is possible to calculate the area, volume and interfacial tension g from a digitized picture of an oil drop in real time using the Laplace formula. The principle is applicable to many different research areas such as enzyme research (kinetics measurements), surfactant or emulsifier development, vegetable oil refining and quality control.

This technique has at Novo Nordisk been found useful for investigating differences in lipase activity towards triolein in buffer at various interfacial tensions. As example are shown the lipase activity towards triolein of Lipolase™ in the absence of calcium at different interfacial tensions.
 
 

Introduction

The oil drop technique is an established method for measuring interfacial tensions at non-miscible liquids interface [1,2]. In the present setup, the oil drop profile is automatically analyzed and the interfacial tension is deduced using the Laplace equation [3]. A barostat has been developed with which it is possible to monitor the enzyme kinetics at a fixed interfacial tension by increasing the drop volume and thereby the drop area [4].

When triolein is hydrolyzed, the free fatty-acids released will lower the surface tension and eventually inhibit the lipase. Different lipases will be influenced differently depending on the interfacial tension level. This technique can be used in the characterization and evaluation of lipases with respect to activity and kinetics in the presence of e.g. surfactants or other surface active molecules.

Materials

The lipase used (E.C. 3.1.1.3) is cloned from Humicula lanuginosa, expressed in Aspergillus oryzae and is commercially available as Lipolase™ (Novo Nordisk). The buffer is a 0.1 M TAPS-buffer at pH=9 (Sigma T-5130) filtered through a 0.45 µm filter to ensure purity. The triglyceride used is triolein (C18:1,[cis]-9, Sigma T-7140), 99% pure. CaCl2 (Merck 2387).

Description of the Oil Drop Tensiometer

The oil drop tensiometer developed by ITConcept [5] is shown in figure 1.

A light source [2] , a cell (cuvette) containing the oil drop [3] , and a CCD camera [6] are aligned on an optical bench [1] . After the drop formation using a syringe [4] and a motor [5] for pressing down the syringe, the drop profile is digitalized [6] through the CCD camera and a personal computer [7] . The monitor [8] is used to align, check, adjust the drop. Two values of area, volume and surface tension are calculated and recorded every second.

Method

Each experiment was initiated by forming a drop of ~40 µl triolein in the cuvette using the computer control, then adding 100 µl 9.15 mg/l lipase to 8 ml buffer in the cuvette. Two types of experiments were carried out. 1. Data of interfacial tension g, area and volume of the drop were recorded with or without 3 mM CaCl2 in the buffer. 2. Data of interfacial tension g, area and volume of the drop were recorded at different levels of interfacial tension, from 11 to 23 mN/m.
 
 

Results

Figure 2 shows the result of having calcium in the buffer when performing the experiment. The decent of the curve representing the interfacial tension is much slower without calcium in the buffer. This could be explained by the lack of formation of calcium soaps during the hydrolysis

Figure 3 shows a typical experiment performed without calcium. The slope of the area-curve representing the growth of the drop (dA/dt) was determined for each experiment and depicted in figure 4.

Figure 4 shows the different dA/dt-values plotted against the selected interfacial tension regulation levels. This curve can be considered to be a unique representation of the lipase in question. In evaluating new lipases, surface activity is considered an important criteria for activity. Using this technique, it is thus possible under realistic conditions to measure the surface activity of the lipase.
 
 

Conclusion

  • The oil drop tensiometer is a valuable tool for determination of interfacial tension between lipid and water and for characterizing lipases with respect to interfacial tension.
  • Calcium effect of the lipase can be investigated easily
  • It is possible to measure lipase activity at a "natural" lipid-water interface independent of the pH, with the possibility of using long chain triglycerides as substrate.
  • By varying the regulation levels of the interfacial tension, it is possible to obtain a unique representation of the activity profile versus the interfacial tension for the lipase in question.
     
References 1. Stauffer CE: J. Phys. Chem. 69(2), 1933-1938, 1965.

2. Patterson E & Ross S: Surface Science 81, 451-463, 1979.

3. Nury S, Piéroni G, Riviére C, Gargouri Y, Bois A & Verger R: Chem. Phys Lipids45, 27-37, 1987.

4. Nury S, Gaudry-Rolland N, Riviére C, Gergouri Y, Bois A, Lin M, Grimaldi M, Richou J & Verger R: In Lipases: Structure, Mechanism & Genetic Engineering, L Alberghina, RD Schmid & R Verger eds. pp. 123-137, VCh., Weinheim, New York. 1991.

5. Cagna A, ITConcept. Rapport Technique Intermediaire Fin de Phase A. Programme Value Oil Drop Tensiometer Contract no. CTT-402. 1993.
6. Cheng P, Li D, Boruvka L, Rotenberg Y & Neumann AW: Colloids and Surfaces 43, 151-167, 1990.



Figure 1. Schematic set-up of the oil drop tensiometer.
 
 
 
 


Figure 2. Experiments showing the difference in interfacial decrease between buffers with or without calcium. Notice the much slower decrease in the buffer without calcium.
 
 
 
 


Figure 3. Typical oil drop tensiometer experiment.
 
 
 
 


 Figure 4. Characteristic curves for Lipolase. Other lipases are considered to have another curve representing their surface activity and can thus be distinguished from Lipolase with respect to surface activity by this method.