3.4.1. Agar-Well Assay Technique
This technique is generally used for screening and is based on diffusion assays in solid medium. The triglycerides emulsified are hydrolyzed by lipases. Lipase activity is monitored by measuring the clearing zone surrounding the wells. The disadvantages of this technique are as follows: (1) tributyrin is not always a good substrate for lipase activity; and (2) the sensitivity of this technique depends on triglyceride concentration. The advantages of this method are as follows: (1) synthetic medium is a reproducible substrate; and (2) it is more adapted for weak lipolytic microorganisms The method is as follows:
1. Emulsify 10 mL of substrate solution at maximal amplitude for 3 min. During sonic treatment, keep cold in an ice-water bath to prevent excessive heating of the emulsion (see Note 7).
2. Add to emulsified substrate solution 1.5 g Agar-Agar ultra pure.
3. Heat emulsion to dissolve the agar
4. Add 15 mL hot emulsion to sterile Petri dishes and solidify.
5. Cut 9-mm-diameter wells in the agar bed.
6. Place 100-^L cellular fractions in the well.
7. Incubate at 30°C (mesophilic bacteria) or 37°C (thermophilic bacteria) for 72 h (see Note 8).
8. The clearing zone surrounding the wells indicates positive lipase activity (see Notes 9 and 10).
In this method, triglycerides are used as the substrate (natural ones such as olive oil or butter fat or synthetic ones such as like triacetin, tributyrin, triolein, and others). Thus, they are often involved in the characterization of lipase, except when triacetin and (less frequently) tributyrin are used. Hydrolysis of triglycerides leads to the production of free fatty acids (FFAs), which can be titrated. Such techniques can be divided into three groups: (1) titration of FFA after extraction; (2) direct titration; and (3) continuous titration. We describe here the titration of FFAs after extraction. The method is as follows:
1. Emulsify 1.5 mL of substrate solution at maximal amplitude for 3 min. During sonic treatment, cool in an ice-water bath to prevent excessive heating of the emulsion
2. Add the emulsified substrate solution to 1 mL cellular fraction, 0.5 mL 10 mM CaCl2/ 1.0 M Tris-HCl buffer, pH 7.0, 0.5 mL 1.0 M NaCl, and 2 mL dH2O (reaction mixture) (see Note 4).
Mix gently and incubate at 30°C (mesophilic bacteria) or 37°C (thermophilic bacteria) for 48 h in shaker (see Notes 11 and 12).
Remove 1 mL of reaction mixture and add 1 mL 0.2 N H2S04/2.0 M NaCl solution for the stop reaction.
Add 6 mL extraction mixture, shake vigorously for 20 s, and let rest for 20 min (see Note 13).
Remove 2 mL of the upper phase, place into an Erlenmeyer flask, add 6 drops of phenol-phthalein, and mix gently.
8. One unit of enzyme activity is defined as the amount of enzyme that released 1 ^mol FFA per min under the conditions of the assay (see Notes 14 and 15).
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