Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis
Abstract
:1. Introduction
2. Results and Discussion
2.1. Analysis of EST5
2.2. Obtaining Protein EST5
2.3. Enzyme Activity and Its Affecting Factors
2.3.1. Effect of pH on Activity and Structure of EST5
2.3.2. Effect of Temperature on the Activity and Stability of EST5
2.3.3. Substrate Specificity and Kinetic Parameters of EST5
2.3.4. Effect of Additives on EST5 Activity
2.4. EST5 Showed Esterification Activity
3. Materials and Methods
3.1. Sequence and Phylogenetic Analysis
3.2. Expression and Purification of the Recombinant Enzyme
3.3. Biophysical Analysis
3.4. Enzyme Characterization
3.4.1. Substrate Selectivity
3.4.2. Effect of pH and Temperature on Enzyme Activity
3.4.3. Effect of Additives on Enzyme Activity
3.5. Esterification Assays
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Substrate | Vmax (µM·s−1) | Km (mM) | Kcat (s−1) | Kcat/Km (s−1 mM−1) |
---|---|---|---|---|
pNP-acetate (C2) | 0.5 ± 0.03 | 1.1 ± 0.13 | 18.97 ± 1.18 | 18.1 |
pNP-butyrate (C4) | 0.78 ± 0.04 | 0.4 ± 0.05 | 29.0 ± 1.46 | 67.7 |
pNP-valerate (C5) | 2.4 ± 0.04 | 0.1 ± 0.01 | 88.9 ± 1.02 | 1533.27 |
pNP-octanoate (C8) | 1.4 ± 0.03 | 0.3 ± 0.04 | 52.2 ± 1.28 | 158.24 |
pNP-decanoate (C10) | 0.3 ± 0.02 | 0.73 ± 0.1 | 12.79 ± 0.74 | 17.5 |
Enzyme | Substrate | Esterification (U·mg−1) | Reference |
---|---|---|---|
EST5 | Butyric acid/Methanol | 127.04 | This study |
LIP4 | Butyric acid/Propanol | 86.5 | [64,65] |
CL | Butyric acid/Propanol | 15.83 | [65] |
LIP2 | Butyric acid/Propanol | 166 | [65] |
Immobilized Lipase PS | Lauric acid/Dodecanol | 8.87 | [66] |
Novozyme 435 | Lauric acid/Dodecanol | 9.89 | [66] |
Lipozyme TLIM | Lauric acid/Dodecanol | 3.54 | [66] |
YLL | Lauric acid/Propanol | 0.35 | [67] |
Sporidiobolus pararoseus lipase | Oleic Acid/Ethanol | 489.65 | [68] |
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Maester, T.C.; Pereira, M.R.; Malaman, A.M.G.; Borges, J.P.; Pereira, P.A.M.; Lemos, E.G.M. Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis. Catalysts 2020, 10, 1100. https://doi.org/10.3390/catal10101100
Maester TC, Pereira MR, Malaman AMG, Borges JP, Pereira PAM, Lemos EGM. Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis. Catalysts. 2020; 10(10):1100. https://doi.org/10.3390/catal10101100
Chicago/Turabian StyleMaester, Thaís Carvalho, Mariana Rangel Pereira, Aliandra M. Gibertoni Malaman, Janaina Pires Borges, Pâmela Aparecida Maldaner Pereira, and Eliana G. M. Lemos. 2020. "Exploring Metagenomic Enzymes: A Novel Esterase Useful for Short-Chain Ester Synthesis" Catalysts 10, no. 10: 1100. https://doi.org/10.3390/catal10101100