Improving Reusability of Biocatalysts by Exploiting Cross-Linked Enzyme Aggregates (CLEAs) with Commercial Cellulolytic Cocktails for Hydrolysis of Green Coconut Waste
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Preparation of Green Coconut Fiber: Alkaline Pre-Treatment with NaOH
2.2.2. Enzyme Cocktails Activity and Protein Concentration
Enzyme Activity Using Filter Paper (FP) as Substrate (FPase)
Enzyme Activity Using Carboxymethyl Cellulose (CMC) as Substrate
2.2.3. Protein Concentration of Enzyme Cocktails
2.2.4. Production of Cross-Linked Enzyme Aggregates (CLEAs)
Selection of Precipitating Agent
Crosslinking of Aggregated Enzymes for the Formation of CLEAs Using Glutaraldehyde
2.2.5. Production of Magnetic Cross-Linked Enzyme Aggregates (m-CLEAs)
Synthesis of Fe3O4 Magnetic Nanoparticles and Coating with 3-Aminopropyltriethoxysilane (APTES)
Formation of m-CLEAs
2.2.6. Biocatalysts Characterization
Immobilization Parameters
Fourier Transform Infrared Spectroscopy (FTIR)
Thermal Stability Assay
SDS-PAGE Electrophoresis
2.2.7. Enzymatic Hydrolysis of Pretreated Biomass
2.2.8. Statistical Analysis
3. Results and Discussion
3.1. Evaluation of Precipitation Agents for the Preparation of the CLEAs
3.2. Evaluation of the Addition of Bovine Serum Albumin (BSA)
3.3. Addition of Magnetic Nanoparticles (m-CLEAs)
3.4. Characterization of m-CLEAs
3.4.1. Variation of Glutaraldehyde Concentration in m-CLEAs
3.4.2. Fourier-Transform Infrared Spectroscopy (FTIR)
3.4.3. SDS-PAGE Electrophoresis
3.4.4. Thermal Inactivation
3.5. Application of m-CLEAs in the Hydrolysis of Pretreated Green Coconut Fiber and Isolated Cellulose
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
APTES | 3-aminopropyltriethoxysilane |
CLEAs | Cross-linked enzyme aggregates |
CMC | Carboxymethyl cellulose |
CMCase | Enzyme activity using carboxymethyl cellulose as substrate |
FP | Filter paper |
FPase | Enzyme activity using filter paper as substrate |
m-CLEAs | Magnetic cross-linked enzyme aggregates |
SDS-PAGE | Sodium dodecyl–sulfate polyacrylamide electrophoresis |
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Precipitating Agent | Celluclast | Cellic CTec2 | ||
---|---|---|---|---|
CMCase | FPase | CMCase | FPase | |
Acetone | 97.83 ± 0.64 | 85.43 ± 1.43 | 91.86 ± 0.13 | 80.23 ± 3.78 |
Ethanol | 98.58 ± 0.69 | 100.00 ± 3.51 | 93.62 ± 0.07 | 88.28 ± 2.92 |
Ammonium sulfate | 100.00 ± 0.04 | 100.00 ± 8.28 | 100.00 ± 0.01 | 100.00 ± 0.98 |
Precipitating Agent | Without BSA | With BSA | ||
---|---|---|---|---|
CMCase | FPase | CMCase | FPase | |
Acetone | 220.10 ± 79.4 | Zero | 287.27 ± 42.59 | Zero |
Ethanol | Zero | Zero | 146.63 ± 13.06 | Zero |
Precipitating Agent | Without BSA | With BSA | ||
---|---|---|---|---|
CMCase | FPase | CMCase | FPase | |
Acetone | 407.06 ± 75.85 | 0.35 ± 0.00 | 425.37 ± 48.11 | 1.54 ± 1.10 |
Ethanol | Zero | Zero | 141.04 ± 26.89 | Zero |
Enzymatic Cocktail | m-CLEAs Activity (U/g) | Yield (%) | ||
---|---|---|---|---|
CMCase | FPase | CMCase | FPase | |
Celluclast | 149.65 ± 2.65 | 5.30 ± 1.75 | 100.00 ± 0.00 | 100.00 ± 0.34 |
Cellic CTec2 | 107.83 ± 21.81 | Zero | 95.55 ± 0.06 | 80.00 ± 3.35 |
CLEAs Activity (U/g) | m-CLEAs–Celluclast 2.5% | m-CLEAs–Celluclast 5.0% |
---|---|---|
CMCase | 299.52 ± 14.28 | 149.65 ± 2.65 |
FPase | 29.52 ± 4.54 | 5.30 ± 1.75 |
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Morais, J.R.F.; Costa, I.O.; Padilha, C.E.A.; Rios, N.S.; Santos, E.S.d. Improving Reusability of Biocatalysts by Exploiting Cross-Linked Enzyme Aggregates (CLEAs) with Commercial Cellulolytic Cocktails for Hydrolysis of Green Coconut Waste. Sustainability 2025, 17, 4221. https://doi.org/10.3390/su17094221
Morais JRF, Costa IO, Padilha CEA, Rios NS, Santos ESd. Improving Reusability of Biocatalysts by Exploiting Cross-Linked Enzyme Aggregates (CLEAs) with Commercial Cellulolytic Cocktails for Hydrolysis of Green Coconut Waste. Sustainability. 2025; 17(9):4221. https://doi.org/10.3390/su17094221
Chicago/Turabian StyleMorais, Jéssica R. F., Isabela O. Costa, Carlos E. A. Padilha, Nathália S. Rios, and Everaldo S. dos Santos. 2025. "Improving Reusability of Biocatalysts by Exploiting Cross-Linked Enzyme Aggregates (CLEAs) with Commercial Cellulolytic Cocktails for Hydrolysis of Green Coconut Waste" Sustainability 17, no. 9: 4221. https://doi.org/10.3390/su17094221
APA StyleMorais, J. R. F., Costa, I. O., Padilha, C. E. A., Rios, N. S., & Santos, E. S. d. (2025). Improving Reusability of Biocatalysts by Exploiting Cross-Linked Enzyme Aggregates (CLEAs) with Commercial Cellulolytic Cocktails for Hydrolysis of Green Coconut Waste. Sustainability, 17(9), 4221. https://doi.org/10.3390/su17094221