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With Gibberella zeae lipase; β‐cyclodextrin In aq. phosphate buffer; chloroform at 25℃; Enzymatic reaction;
2.3 Enzymatic activity determination
General procedure: Lipolytic activity of wild-type GZEL and its two mutants to various substrates was performed using the emulsion system and assayed with a pH-stat apparatus (Radiometer, Copenhagen, Denmark) according to a reported procedure [19]. Olive oil and soybean phospholipid were used as substrate to test the lipase activity and phospholipase activity, respectively. Specific activity was expressed in units (U) per gram of enzyme. One U corresponds to the amount of enzyme that releases 1μmol of free fatty acid per minute. (0009) The hydrolysis of different phospholipid or galactolipid monomolecular film by GZEL and its mutants were followed by measuring the simultaneous decrease with time in the film area using a “zero-order” trough at a constant surface pressure of 25 mN m-1. The “zero-order” trough from Kibron (Helsinki, Finland) is composed of one reaction compartment (volume 2.5 mL, surface area 3.8 cm2) and two reservoir compartments (volume 27.7 mL, surface area 55.5 cm2 for each side) connected to each other by a small surface channel. Experiments were performed at room temperature (25°C). Herein, hydrolytic activities were tested under pH value of 6.0. This value was found to be the optimum pH condition for the phospholipase activity of GZEL and its two truncation mutants that tested under emulsion system. Monolayer was prepared by spreading a few microliters of phospholipid solution in chloroform on the 50mMPB buffer (pH6.0) subphase. A mobile barrier, automatically driven by the barostat, moved back and forth over the reservoir to compress and keep the surface pressure constant at 25mNm-1. In order to ensure that the long chain fatty acid produced in the hydrolytic reaction could solubilize in the water and thus allows to leave space for new incoming lipids from the reservoir, β-Cyclodextrin (final bulk concentration of 3mgmL-1 on the subphase PB buffer) was included in the reaction compartment. The principle of this method has been previously described [20]. Then the enzyme solution was injected into the subphase of the reaction compartment to start the reaction. A magnetic stirrer (diameter 0.5cm) was used to stir the subphase at 250rpm. Activities were expressed as the number of moles of substrate hydrolyzed per unit time and unit reaction compartment area per milligram of enzyme in the “zero-order” trough (moles·cm-2·min-1·mg-1). Values are presented as mean±standard deviation based on three independent experiments.
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