Preparation and Biochemical Characterization of Penicillium crustosum Thom P22 Lipase Immobilization Using Adsorption, Encapsulation, and Adsorption-Encapsulation Approaches

Abstract

This work describes the immobilization and the characterization of purified Penicillium crustosum Thom P22 lipase (PCrL) using adsorption, encapsulation, and adsorption-encapsulation approaches. The maximum activity of the immobilized PCrL on CaCO₃ microspheres and sodium alginate beads was shifted from 37 to 45 °C, compared with that of the free enzyme. When sodium alginate was coupled with zeolite or chitosan, the immobilization yield reached 100% and the immobilized PCrL showed improved stability over a wide temperature range, retaining all of its initial activity after a one-hour incubation at 60 °C. The immobilization of PCrL significantly improves its catalytic performance in organic solvents, its pH tolerance value, and its thermal stability. Interestingly, 95% and almost 50% of PCrL's initial activity was retained after 6 and 12 cycles, respectively. The characteristics of all PCrL forms were analyzed by X-ray diffraction and scanning electron microscopy combined with energy dispersive spectroscopy. The maximum conversion efficiency of oleic acid and methanol to methyl esters (biodiesel), by PCrL immobilized on CaCO₃, was 65% after a 12 h incubation at 40 °C, while free PCrL generated only 30% conversion, under the same conditions.

Keywords: Penicillium crustosum lipase; esterification; hybrid carriers; immobilization.

Figure 1

Adsorption kinetics of PCrL on CaCO₃ and Celite 545. PCrL adsorbed on CaCO₃ leads to the highest yield of 90% after 30 min of incubation (4500 U) at 4 °C. The lipase activity was measured with the pH-STAT technique using TC8 as the substrate.

Figure 2

Identification of the PCrL-SA-ZE, PCrL-SA-CS, and PCrL-CaCO₃ beads. (a) The XRD patterns of the support beads alone (SA, SA-ZE, or SA-CS) or complexed with PCrL (PCrL-SA, PCrL-SA-ZE, and PCrL-SA-CS). The XRD patterns were generated using Match! software (version 3.10.2.173); (b) Identification of the support beads (SA, SA-CS, and SA-ZE) and those complexed with PCrL (PCrL-SA, PCrL-SA-CS, and PCrL-SA-ZE) in the FE-SEM images.

Figure 3

The effect of temperature on PCrL activity and stability. (a) The temperature–activity profile. This graph shows the activity of the free and immobilized PCrL at different temperatures. The activity of the immobilized PCrL at its optimal temperature (45 °C) is set at 100%. (b) Temperature stability. This graph illustrates the stability of the free and immobilized PCrL after incubation at different temperatures for 60 min. The residual activity was measured at pH value 9 using TC8 as a substrate. Each data point on the graph represents the average of three independent experiments.

Figure 4

The effect of pH on PCrL activity and stability. (a) The pH activity profile of the free and immobilized PCrL at different pH values. The maximum activity at pH value 9 is set at 100%. (b) pH value stability of the free and immobilized PCrL after incubation at different pH values for 1 h at 4 °C. The residual activity was measured at pH value 9 and 37 °C using TC8 as the substrate. Each data point represents the average of three independent experiments.

Figure 5

The effect of organic solvents on PCrL-CaCO₃ activity and stability. The enzyme was incubated with 25% (v/v) of each solvent for 24 h. The residual activity was measured under standard conditions, using TC8 as the substrate at 37 °C and pH 9, as described in Material and Methods, and then expressed as a percentage of the activity without any solvents. Each data point represents the average of three independent experiments, with the error bars indicating standard deviation.

Figure 6

Performance evaluation of PCrL-CaCO₃. (a) The kinetics of oleic acid esterification catalyzed by the free PCrL and immobilized PCrL-CaCO₃. The reaction was performed at 40 °C, with stirring for 24 h, using 500 U of enzyme in hexane with a 3:1 molar ratio of methanol to oleic acid. (b) The reusability of immobilized PCrL-CaCO₃ in multiple reaction cycles. The enzyme was reused for 12 cycles, with each cycle lasting 12 h. The conversion yield of oleic acid to esters was monitored for each cycle.

Figure 7

The thermodynamic parameters of the target PCrL. An Arrhenius diagram of Ln (k_d) vs. 1/temperature to calculate the activation energy (E_a).