Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization
Abstract
:1. Introduction
2. Results and Discussion
2.1. Cloning and Production of ARA-1 and ARA-2
2.2. Purification of ARA-1 and ARA-2
2.3. Physicochemical Properties of ARA-1 and ARA-2
2.4. Substrate Specificity and Kinetics of ARA-1 and ARA-2
2.5. Cellulose-Binding Assay
2.6. Synergistic Effect of Arabinofuranosidases with Endoxylanase in the Production of XOS
2.7. Role of ARA-1 and ARA-2 in Saccharification Processes
3. Materials and Methods
3.1. Nucleic Acid Isolation and Cloning of Two α-l-Arabinofuranosidases
3.2. Production and Purification of ARA-1 and ARA-2
3.3. Determination of the Physicochemical Properties of the Recombinant ARA-1 and ARA-2
3.4. Substrate Specificity of Recombinant ARA-1 and ARA-2
3.5. XOS Release from Arabinoxylan and Saccharification
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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ARA-1 | |||
---|---|---|---|
Step | Specific Activity (U/g) | Yield (%) | Purification Fold |
Crude extracts | 16.9 | 100 | - |
Purified enzyme | 67.5 | 73 | 4 |
ARA-2 | |||
Step | Specific Activity (U/g) | Yield (%) | Purification Fold |
Crude extracts | 200 | 100 | - |
Purified enzyme | 890 | 71.8 | 4.5 |
Organism | Enzyme | Optimum pH | pH Stability | Optimum Temperature | Temperature Stability | Wheat Arabinoxylan | pNP-AF | Arabinan |
---|---|---|---|---|---|---|---|---|
kcat/Km (s−1 mg−1 mL) | Specific Activity (U/mg) | Specific Activity (U/mg) | ||||||
Eupenicillium parvum [16] | EpABF62A | 4.5 | 2–11 | 55 | 50 (24 h) | 12 | 0.4 | - |
Aspergillus nidulans [17] | AnAbf62A-m2,3 | 5.5 | - | 70 | - | 36.3 | 1.66 | 1.43 |
Streptomyces sp. SWU10 [18] | WUAbf62A | 5 | 4–9 | 50 | 50 (1 h) | 0.15 * | - | 0.01 * |
Nectria haematococca [19] | NhaAbf62A | 6.5 | - | 40 | 40 (45 min) | 6.1 | - | 0.4 |
Sporisorium reilianum [19] | SreAbf62A | 6.5 | - | 40 | 50 (45 min) | 3.3 | - | 0 |
Gibberella zeae [19] | GzeAbf62A | 6.5 | - | 40 | 40 (45 min) | 8.2 | - | 0 |
Penicillium funiculosum [20] | ABF62a | 3.5 | - | 40 | - | 7.25 | - | - |
ABF62b | 2.2 | - | 40 | - | 153.6 | - | - | |
ABF62c | 3 | - | 50 | - | 55.1 | - | - | |
Penicillium subrubescens [21] | AxhA | 5 | 4–8 | 40 | 60 (1 h) | - | 4.1 | - |
AxhB | 5 | 3–8 | 40 | 50 (1 h) | - | 14.6 | - | |
AxhC | 5 | 3–7 | 40 | 50 (1 h) | - | 3.8 | - | |
AxhD | 4 | 2–7 | 40 | 50 (1 h) | - | 10.9 | - | |
Scytalidium thermophilum [22] | Abf62A | 4–5 | - | 50 | - | 5.5 | 0.02 | |
Abf62C | 6–7 | - | 50 | - | 4.5 | 0.24 | ||
Talaromyces amestolkiae (this work) | ARA-1 | 4 | 3–7 | 50 | 40 (72 h) | 129 | 0.07 | 0.3 |
ARA-2 | 4 | 2–9 | 60 | 50 (72 h) | 9.60 | 0.5 | 0.28 |
Substrate | ARA-1 | ARA-2 | |
---|---|---|---|
pNP-AF | Km (mM) | 8.97 ± 0.06 | 1.48 ± 0.08 |
kcat (s−1) | 0.05 ± 0.001 | 0.41 ± 0.01 | |
kcat/Km (mM−1·s−1) | 0.005 ± 0.0004 | 0.28 ± 0.01 | |
Arabinoxylan | Km (mg/mL) | 0.29 ± 0.11 | 1.80 ± 0.43 |
kcat (s−1) | 37.17 ± 1.97 | 16.89 ± 1.23 | |
kcat/Km (mg−1·mL·s−1) | 129.2 ± 3.2 | 9.60 ± 0.27 | |
Arabinan | Km (mg/mL) | 1.82 ± 0.23 | 3.4 ± 0.42 |
kcat (s−1) | 0.20 ± 0.01 | 0.23 ± 0.01 | |
kcat/Km (mg−1·mL·s−1) | 0.11 ± 0.001 | 0.07 ± 0.01 |
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Méndez-Líter, J.A.; de Eugenio, L.I.; Nieto-Domínguez, M.; Prieto, A.; Martínez, M.J. Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization. Int. J. Mol. Sci. 2023, 24, 11997. https://doi.org/10.3390/ijms241511997
Méndez-Líter JA, de Eugenio LI, Nieto-Domínguez M, Prieto A, Martínez MJ. Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization. International Journal of Molecular Sciences. 2023; 24(15):11997. https://doi.org/10.3390/ijms241511997
Chicago/Turabian StyleMéndez-Líter, Juan A., Laura I. de Eugenio, Manuel Nieto-Domínguez, Alicia Prieto, and María Jesús Martínez. 2023. "Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization" International Journal of Molecular Sciences 24, no. 15: 11997. https://doi.org/10.3390/ijms241511997
APA StyleMéndez-Líter, J. A., de Eugenio, L. I., Nieto-Domínguez, M., Prieto, A., & Martínez, M. J. (2023). Expression and Characterization of Two α-l-Arabinofuranosidases from Talaromyces amestolkiae: Role of These Enzymes in Biomass Valorization. International Journal of Molecular Sciences, 24(15), 11997. https://doi.org/10.3390/ijms241511997