Improved Cellulolytic Activity of Alternaria citri: Optimization and EMS Treatment for Enhanced Cellulase Production
Abstract
:1. Introduction
2. Materials and Methods
2.1. Microorganism and Culture Conditions
2.2. Screening of A. citri Cellulases
2.3. Chemical Mutagenesis Using EMS
2.4. Screening and Selection of Mutant Strains
2.4.1. Endoglucanase Assay
2.4.2. Exoglucanase Assay
2.4.3. β-Glucosidase
2.5. Optimization of Physical Factors for Cellulase Production
2.6. Optimization of Nutritional Requirements for Cellulase Production
2.7. Cellulase Purification from Wild A. citri and Mutant A. citri 305
2.8. Determination of Protein Concentration
2.9. Enzyme Characterization from Wild A. citri and Mutant A. citri 305
2.9.1. Effects of Temperature, pH, Metal Ions, and Additives on Purified Cellulase Activity
2.9.2. Enzyme Kinetics of Purified Cellulases from Wild A. citri and Mutant A. citri 305
2.10. Statistical Analysis
3. Results and Discussion
3.1. Effects of EMS Mutagenesis and Cellulolytic Screening of Mutants
3.2. Optimization of Physical Parameters for Cellulase Production by Wild A. citri and Mutant A. citri 305
3.2.1. Temperature Optimization
3.2.2. Optimization of pH
3.2.3. Incubation Period Optimization
3.3. Optimization of Nutritional Requirements for Cellulase Production by A. citri and A. citri 305
3.3.1. Substrate Optimization
3.3.2. Carbon Source Optimization
3.3.3. Nitrogen Source Optimization
3.4. Cellulase Purification from Wild A. citri and Mutant Strain A. citri 305
3.5. Characterization of Purified Cellulases from Wild A. citri and Mutant Strain A. citri 305
3.5.1. Effect of Temperature on the Activity of Purified Cellulases
3.5.2. Effect of pH on Purified Cellulase Activity
3.5.3. Effects of Metal Ions on Purified Cellulase Activity
3.5.4. Thermo-Stability Studies on Cellulases from Wild A. citri and Mutant Strain A. citri 305
3.5.5. pH Stability Studies on Purified Cellulases from Wild A. citri and Mutant Strain of A. citri 305
3.6. Kinetics Analysis of Purified Cellulase
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
EMS | Ethyl methanesulfonate |
CMC | Carboxymethyl cellulose |
OP | Orange peel |
SB | Sugarcane bagasse |
WS | Wheat straw |
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Cellulases | Parameters | Culture Supernatant | Partially Purified Enzyme | ||
A. citri | A. citri 305 | A. citri | A. citri 305 | ||
TP (mg/mL) | 1.07 ± 0.02 | 1.55 ± 0.01 | 0.35 ± 0.01 | 0.17 ± 0.02 | |
Endoglucanase | TA (U) | 0.43 ± 0.03 | 0.65 ± 0.04 | 0.27 ± 0.01 | 0.28 ± 0.01 |
SA (U/mg) | 0.40 ± 0.03 | 0.42 ± 0.026 | 0.85 ± 0.03 | 1.65 ± 0.18 | |
PF | 1 | 1 | 2.12 | 3.95 | |
R (%) | 100 | 100 | 62.79 | 43.07 | |
Exoglucanase | TA (U) | 0.26 ± 0.02 | 0.27 ± 0.05 | 0.09 ± 0.003 | 0.10 ± 0.01 |
SA (U/mg) | 0.24 ± 0.02 | 0.18 ± 0.034 | 0.28 ± 0.02 | 0.58 ± 0.06 | |
PF | 1 | 1 | 1.17 | 3.34 | |
R (%) | 100 | 100 | 34.61 | 37.03 | |
β-glucosidase | TA (U) | 8.59 ± 1.44 | 12.85 ± 0.91 | 3.88 ± 0.31 | 1.49 ± 0.26 |
SA (U/mg) | 8.02 ± 1.35 | 8.29 ± 0.588 | 12.26 ± 1.05 | 8.76 ± 1.79 | |
PF | 1 | 1 | 1.52 | 1.05 | |
R (%) | 100 | 100 | 45.16 | 11.59 |
Metal Ions (10%) | Relative Activity | Relative Activity | Relative Activity | |||
---|---|---|---|---|---|---|
(%) | (%) | (%) | ||||
β-glucosidase | Endoglucanase | Exoglucanase | ||||
A. citri | A. citri 305 | A. citri | A. citri 305 | A. citri | A. citri 305 | |
Control | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 |
NaNO3 | 104 ± 1 | 103 ± 1 | 96 ± 1 | 96 ± 1 | 126 ± 1 | 104 ± 3 |
FeSO4 | 127 ± 1 | 139 ± 3 | 203 ± 1 | 137 ± 1 | 275 ± 1 | 285 ± 2 |
MgSO4 | 107 ± 1 | 135 ± 2 | 119 ± 2 | 113 ± 1 | 135 ± 2 | 205 ± 2 |
ZnSO4 | 120 ± 1 | 108 ± 2 | 131 ± 2 | 112 ± 1 | 156 ± 0 | 195 ± 2 |
KH2PO4 | 79 ± 3 | 90 ± 4 | 96 ± 1 | 94 ± 0 | 115 ± 1 | 118 ± 0 |
CoCl2 | 53 ± 1 | 73 ± 3 | 70 ± 1 | 73 ± 1 | 95 ± 1 | 78 ± 3 |
Additives (5 mM) | β-glucosidase | Endoglucanase | Exoglucanase | |||
(%) | (%) | (%) | ||||
A. citri | A. citri 305 | A. citri | A. citri 305 | A. citri | A. citri 305 | |
Control | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 | 100 ± 0 |
SDS | 56 ± 2 | 96 ± 1 | 63 ± 1 | 60 ± 1 | 41 ± 2 | 54 ± 1 |
Tween 80 | 107 ± 2 | 108 ± 0 | 118 ± 2 | 112 ± 1 | 126 ± 2 | 165 ± 1 |
EDTA | 67 ± 3 | 97 ± 1 | 77 ± 1 | 83 ± 1 | 78 ± 2 | 90 ± 1 |
Urea | 68 ± 3 | 59 ± 2 | 96 ± 3 | 75 ± 0 | 86 ± 2 | 64 ± 4 |
Cellulases | Strains | Km | Vmax | Efficiency |
---|---|---|---|---|
(mg/mL) | (mg/mL/min) | (/min) | ||
Endoglucanase | A. citri | 0.492 ± 0.004 | 0.039 ± 0.000 | 0.078 ± 0.001 |
A. citri 305 | 0.295 ± 0.006 | 0.060 ± 0.000 | 0.202 ± 0.004 | |
Exoglucanase | A. citri | 0.278 ± 0.011 | 0.023 ± 0.000 | 0.084 ± 0.003 |
A. citri 305 | 0.241 ± 0.007 | 0.023 ± 0.000 | 0.094 ± 0.004 | |
β-glucosidase | A. citri | 0.048 ± 0.001 | 0.754 ± 0.011 | 15.683 ± 0.099 |
A. citri 305 | 0.031 ± 0.001 | 1.128 ± 0.004 | 36.765 ± 1.598 |
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Ahmed, S.; Andaleeb, H.; Aslam, A.; Raza, J.A.; Waseem, S.M.Y.; Javaid, A.; Talib, C. Improved Cellulolytic Activity of Alternaria citri: Optimization and EMS Treatment for Enhanced Cellulase Production. Fermentation 2025, 11, 274. https://doi.org/10.3390/fermentation11050274
Ahmed S, Andaleeb H, Aslam A, Raza JA, Waseem SMY, Javaid A, Talib C. Improved Cellulolytic Activity of Alternaria citri: Optimization and EMS Treatment for Enhanced Cellulase Production. Fermentation. 2025; 11(5):274. https://doi.org/10.3390/fermentation11050274
Chicago/Turabian StyleAhmed, Sibtain, Hina Andaleeb, Aqsa Aslam, Junaid Ahmad Raza, Sheikh Muhammad Yahya Waseem, Atayyaba Javaid, and Chand Talib. 2025. "Improved Cellulolytic Activity of Alternaria citri: Optimization and EMS Treatment for Enhanced Cellulase Production" Fermentation 11, no. 5: 274. https://doi.org/10.3390/fermentation11050274
APA StyleAhmed, S., Andaleeb, H., Aslam, A., Raza, J. A., Waseem, S. M. Y., Javaid, A., & Talib, C. (2025). Improved Cellulolytic Activity of Alternaria citri: Optimization and EMS Treatment for Enhanced Cellulase Production. Fermentation, 11(5), 274. https://doi.org/10.3390/fermentation11050274