Preparation and Biochemical Characterization of Penicillium crustosum Thom P22 Lipase Immobilization Using Adsorption, Encapsulation, and Adsorption–Encapsulation Approaches
<p>Adsorption kinetics of PCrL on CaCO<sub>3</sub> and Celite 545. PCrL adsorbed on CaCO<sub>3</sub> 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.</p> "> Figure 2
<p>Identification of the PCrL-SA-ZE, PCrL-SA-CS, and PCrL-CaCO<sub>3</sub> beads. (<b>a</b>) 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>b</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.</p> "> Figure 3
<p>The effect of temperature on PCrL activity and stability. (<b>a</b>) 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>b</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.</p> "> Figure 4
<p>The effect of pH on PCrL activity and stability. (<b>a</b>) 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>b</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.</p> "> Figure 5
<p>The effect of organic solvents on PCrL-CaCO<sub>3</sub> activity and stability. The enzyme was incubated with 25% (<span class="html-italic">v</span>/<span class="html-italic">v</span>) 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.</p> "> Figure 6
<p>Performance evaluation of PCrL-CaCO<sub>3</sub>. (<b>a</b>) The kinetics of oleic acid esterification catalyzed by the free PCrL and immobilized PCrL-CaCO<sub>3</sub>. 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>b</b>) The reusability of immobilized PCrL-CaCO<sub>3</sub> 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.</p> "> Figure 7
<p>The thermodynamic parameters of the target PCrL. An Arrhenius diagram of Ln (<span class="html-italic">k</span><sub>d</sub>) vs. 1/temperature to calculate the activation energy (E<sub>a</sub>).</p> ">
Abstract
:1. Introduction
2. Results
2.1. Immobilization of PCrL and the Characterization of Different Forms
2.2. XRD Analysis of Immobilized PCrL
2.3. FE-SEM Analysis of Immobilized PCrL
2.4. Biochemical Characterization of Immobilized PCrL
2.5. Evaluation of the Performance of Immobilized PCrL in Organic Synthesis
2.6. Thermodynamic Parameters of the Thermal Inactivation of PCrL
3. Discussion
4. Material and Methods
4.1. Material
4.2. Lipase Activity Measurements and Protein Determination
4.3. PCrL Immobilization
4.4. Characterization of PCrL Beads
4.5. Biochemical Characterization of Immobilized PCrL
4.6. Performance of Immobilized PCrL in Organic Synthesis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Methods | PCrL-Support | Initial Enzyme Activity 1 (U/g of Ball) | Unbound Enzyme Activity (U/g of Ball) | Bound Enzyme Activity (U/g of Ball) | Immobilization Yield (%) | Activity Recovered (%) |
---|---|---|---|---|---|---|
Encapsulation | SA (2%) | 40 | 2.35 | 7.5 | 94 | 19.9 |
SA (2%)-CS (0.8%) | 45 | 1.85 | 11.7 | 95 | 27.2 | |
Encapsulation–Adsorption | SA (2%)-ZE (1.2%) | 37 | 0 | 17.5 | 100 | 47.3 |
Enzyme | Temperature (K) | t1/2 (h) | kd (h−1) | D (h) | ΔHd (kJ·mol−1) | ΔGd (J·mol−1) | ΔS (J·mol−1 K−1) | Ed (kJ·mol−1) | Ea (kJ·mol−1) |
---|---|---|---|---|---|---|---|---|---|
Free PCrL | 313 | 1 | 0.346 | 6.656 | −16.80 | 2 | −53.68 | −7.28 | −0.60 |
323 | 0.5 | 0.693 | 3.323 | −17.10 | −1.12 | −52.93 | |||
333 | 0.25 | 0.326 | 7.064 | −17.41 | −1.16 | −52.27 | |||
PCrL-SA | 313 | 4 | 0.173 | 13.312 | −13.16 | −1.08 | −42.04 | −3.64 | −0.03 |
323 | 3 | 0.231 | 10.013 | −13.46 | 2.85 | −41.68 | |||
333 | 2 | 0.346 | 6.656 | −13.77 | 1.16 | −41.35 | |||
PCrL-CaCO3 | 313 | 6 | 0.115 | 13.312 | −13.18 | −1.15 | −42.10 | −3.66 | −0.033 |
323 | 4 | 0.173 | 13.312 | −13.48 | −1.16 | −41.73 | |||
333 | 3 | ar0.231 | 10.013 | −13.77 | −1.16 | −41.34 |
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Hasnaoui, I.; Mechri, S.; Dab, A.; Bentouhami, N.E.; Abouloifa, H.; Bellaouchi, R.; Allala, F.; Saalaoui, E.; Jaouadi, B.; Noiriel, A.; et al. Preparation and Biochemical Characterization of Penicillium crustosum Thom P22 Lipase Immobilization Using Adsorption, Encapsulation, and Adsorption–Encapsulation Approaches. Molecules 2025, 30, 434. https://doi.org/10.3390/molecules30030434
Hasnaoui I, Mechri S, Dab A, Bentouhami NE, Abouloifa H, Bellaouchi R, Allala F, Saalaoui E, Jaouadi B, Noiriel A, et al. Preparation and Biochemical Characterization of Penicillium crustosum Thom P22 Lipase Immobilization Using Adsorption, Encapsulation, and Adsorption–Encapsulation Approaches. Molecules. 2025; 30(3):434. https://doi.org/10.3390/molecules30030434
Chicago/Turabian StyleHasnaoui, Ismail, Sondes Mechri, Ahlem Dab, Nour Eddine Bentouhami, Houssam Abouloifa, Reda Bellaouchi, Fawzi Allala, Ennouamane Saalaoui, Bassem Jaouadi, Alexandre Noiriel, and et al. 2025. "Preparation and Biochemical Characterization of Penicillium crustosum Thom P22 Lipase Immobilization Using Adsorption, Encapsulation, and Adsorption–Encapsulation Approaches" Molecules 30, no. 3: 434. https://doi.org/10.3390/molecules30030434
APA StyleHasnaoui, I., Mechri, S., Dab, A., Bentouhami, N. E., Abouloifa, H., Bellaouchi, R., Allala, F., Saalaoui, E., Jaouadi, B., Noiriel, A., Asehraou, A., & Abousalham, A. (2025). Preparation and Biochemical Characterization of Penicillium crustosum Thom P22 Lipase Immobilization Using Adsorption, Encapsulation, and Adsorption–Encapsulation Approaches. Molecules, 30(3), 434. https://doi.org/10.3390/molecules30030434