Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases
Abstract
:1. Introduction
2. Results
2.1. Water Sampling
2.2. Library Construction
2.3. Functional Screening
2.3.1. Screening for Plant Cell Wall Degrading Enzymes
2.3.2. Screening for Proteases
2.3.3. Screening for Lipolytic and Feruloyl Esterase Activities
2.4. Subcloning of Selected Positive Lypolitic Activity-Conferring Genes and Sequence Analysis
2.5. Alignment of LipB12_A11 with NCBI Databases for DNA and Protein Sequences
2.6. Purification and Biochemical Characterization of Lipolytic Activity Enzyme LipB12_A11
2.6.1. LipB12_A11 Temperature and pH Optima and Thermostability
2.6.2. LipB12_A11 Substrate Preference
2.6.3. Effect of Additives and Detergents on LipB12_A11 Enzymatic Activity
2.6.4. LipB12_A11 Enzyme Kinetics
3. Discussion
4. Materials and Methods
4.1. Sampling
4.2. DNA Extraction
4.3. Metagenomic Libraries Construction
4.3.1. End Repair and Size Selection of Metagenomic DNA
4.3.2. Vector
4.3.3. Ligation and Packaging of Metagenomic DNA
4.4. Functional Screening Methods
4.4.1. Cellulase Screening
4.4.2. Xylanase Screening
4.4.3. β-Xylosidase Screening
4.4.4. Lipolytic Activity Screening
4.4.5. Feruloyl Esterase Screening
4.4.6. β-Glucosidase Screening
4.4.7. Protease Screening
4.5. Subcloning of a DNA Fragment Containing a Lipolytic Activity-Conferring Gene
4.6. Sequencing and Primer Walking of a DNA Fragment Containing a Lipolytic Activity-Conferring Gene
4.7. Sequence Analysis
4.8. Purification
4.9. Activity Assays
5. 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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Enzymatic Activity | Substrate | Method | As Burgas | Muiño da Veiga |
---|---|---|---|---|
Endoglucanase | CMC | Halo | 0 | 0 |
AZCL-HE-Celulose | Colorimetric | 0 | 0 | |
EnzChek cellulase | Fluorimetric | 2 | 4 | |
Exoglucanase | AVICEL | Halo | 0 | 0 |
β-glucosidase | pNP-glucopyranoside | Colorimetric | 5 | 1 |
Xylanase | AZCL-Xylan | Colorimetric | 0 | 0 |
β-xylosidase | MUX | Fluorimetric | 2 | 0 |
Protease | EnzChek Casein | Fluorimetric | 4 | 6 |
Lipolytic | Tributyrin | Halo | 1 | 8 |
Feruloyl esterase | MUTMAC | Fluorimetric | 0 | 0 |
Purification Step | Volume (mL) | Total Activity (U/µL) | Total Protein (mg) | Specific Activity (U/mg) | Yield (%) | Purification Fold |
---|---|---|---|---|---|---|
Crude extract | 16 | 0.000201 | 17.01 | 1.1807 × 10−8 | 100 | 1 |
Differential thermal precipitation | 8 | 0.000155 | 10.88 | 1.4287 × 10−8 | 77.42 | 1.21 |
Ammonium sulphate precipitation | 8 | 0.000149 | 4.33 | 3.4641 × 10−8 | 74.69 | 2.93 |
Molecular exclusion chromatography | 3 | 0.000145 | 2.70 | 5.383 × 10−8 | 72.49 | 4.56 |
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Escuder-Rodríguez, J.-J.; DeCastro, M.-E.; Saavedra-Bouza, A.; González-Siso, M.-I.; Becerra, M. Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases. Int. J. Mol. Sci. 2022, 23, 5733. https://doi.org/10.3390/ijms23105733
Escuder-Rodríguez J-J, DeCastro M-E, Saavedra-Bouza A, González-Siso M-I, Becerra M. Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases. International Journal of Molecular Sciences. 2022; 23(10):5733. https://doi.org/10.3390/ijms23105733
Chicago/Turabian StyleEscuder-Rodríguez, Juan-José, María-Eugenia DeCastro, Almudena Saavedra-Bouza, María-Isabel González-Siso, and Manuel Becerra. 2022. "Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases" International Journal of Molecular Sciences 23, no. 10: 5733. https://doi.org/10.3390/ijms23105733
APA StyleEscuder-Rodríguez, J.-J., DeCastro, M.-E., Saavedra-Bouza, A., González-Siso, M.-I., & Becerra, M. (2022). Bioprospecting for Thermozymes and Characterization of a Novel Lipolytic Thermozyme Belonging to the SGNH/GDSL Family of Hydrolases. International Journal of Molecular Sciences, 23(10), 5733. https://doi.org/10.3390/ijms23105733