Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Two Esterase-Positive Clones from the Oil-Polluted Mud Flat Metagenome Library
2.3. Sequence Analysis and Phylogenetic Tree
2.4. Enzyme Assays
2.5. Preparation of Crude Enzymes
2.6. Isolation of the Enzymes
2.7. Characterization of the Enzymes
3. Results
3.1. Sequence Analyses and Multiple Alignments of Est2L and Est4L
3.2. Isolation of Est2L and Est4L
3.3. Propeties of Est2L and Est4L
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Enzyme | Preparation | Specific Activity (U/mg) | Purification (Fold) | Yield (%) |
---|---|---|---|---|
Est2L | Crude extract | 0.043 | 1.00 | 100 |
HiTrap-Q | 0.11 | 2.56 | 22.6 | |
Crude extract | 0.041 | 1.00 | 100 | |
Sephacryl S-200 | 0.22 | 5.36 | 9.45 | |
Est4L | Crude extract | 0.037 | 1.00 | 100 |
HiTrap-Q | 0.063 | 1.70 | 12.5 | |
Sephacryl S-200 | 0.027 | 0.73 | 2.25 |
Protein | Fraction Volume (mL) | Molecular Mass (kDa) | log Mw |
---|---|---|---|
Est2L | 37.5 | 183.2 ± 23.6 | 2.26 |
Est4L | 43.6 | 125.4 ± 8.65 | 2.10 |
IgG | 39 | 166 | 2.22 |
BSA | 54 | 66.4 | 1.82 |
Protein | Accession | Source | Homology (%) | AA | MW (kDa) | Native Form | Opt Temp. (°C) | Opt. pH | pNP Esters | Solvent Stability (%) | Metal Ion | Ref. | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
IPA | MeOH | ACN | Activating | Inhibiting | |||||||||||
Family IV | |||||||||||||||
Est2L * | MT989338 | Uncultured bacterium | 100 | 839 | 92.5 | Di | 60 | 10.0 | C2 | 15.7 a | 92.7 a | 6.67 a | Mg2+, Mn2+, Fe2+ | Cu2+ | This study |
Est3K | AKG92633 | Uncultured bacterium | 19.90 | 299 | 32.4 | 50 | 9 | C4 | 87.5 a | 86.9 a | 47.7 a | Cu2+ | [29] | ||
EstE7 | ABI18352 | Uncultured bacterium | 27.93 | 322 | 34.6 | Di | 40 | 5 | C4 | [47] | |||||
Cest-2923 | CCC79999 | Lactobacillus plantarum | 9.75 | 282 | 31.2 | Di, Tetra | 30 | 7 | C2 | [48] | |||||
REst1 | FJ645062 | Rheinheimera sp. | 15.56 | 342 | 37.2 | Mono, Tri | 50 | 8 | C4 | 0 a | Cd2+ | Mg2+, Hg2+ | [49] | ||
E69 | AUD08548 | Erythrobacter seohaensis | 18.18 | 274 | 29.5 | 10.5 | 6 | C4 | 0 b | 28.8 b | 35.0 b | [53] | |||
EstE1 | AAW62260 | Uncultured archaeon | 19.85 | 311 | 33.7 | Di | 95 | 6 | C6 | [54] | |||||
Est22 | AFB82695 | Uncultured bacterium | 16.51 | 365 | 39 | Di | 40 | 7.5 | C2 | 40 b | 120 b | 0 b | Zn2+, Cu2+, Ni2+ | [57] | |
EstA1 ** | PHS53692.1 | Alteromonas sp. | 18.97 | 379 | 41.2 | 45 | 8 | C2 | 102 b | 100 b | 68 b | [58] | |||
PestE | BAC06606 | Pyrobaculum calidifontis | 20.49 | 313 | 34.4 | Di | 90 | 7 | C6 | 110 c | 109 c | 117 c | [59,61] | ||
EST2 | QGT40748 | Alicyclobacillus acidocaldarius | 20.34 | 311 | 34.4 | Mono | 70 | 7.1 | C6 | [60] | |||||
Family V | |||||||||||||||
Est4L | MT989339 | Uncultured bacterium | 100 | 267 | 30.1 | Tetra | 50 | 10.0 | C10 | 6.94 a | 128.6 a | 7.47 a | Cu2+, Zn2+ | This study | |
Est16 | ADM63076 | Uncultured bacterium | 21.28 | 302 | 31.9 | Mono | 55 | 9.0 | C4 | Mg2+, Mn2+ | Zn2+ | [50] | |||
QpeH | ANT80587 | Pseudomonas sp. J-5 | 17.67 | 309 | 38.5 | Mono | 30 | 8.0 | Ca2+, Li2+, Cd2+ | Mg2+ | [51] | ||||
LacH | AGS18892 | Brevundimonas sp. LY-2 | 12.46 | 306 | 32.4 | Di | 40 | 7.0 | C2 | Fe2+, Cd2+ | Zn2+, Hg2+ | [52] | |||
H8 | ARH02619 | Uncultured bacterium | 17.33 | 305 | 32.8 | 35 | 10.8 | C6 | 4.4 d | 52.6 d | 2.7 d | Cu2+, Zn2+ | [55] | ||
FCLip1 | ABL95965 | Fervidobacterium changbaicum | 10.64 | 315 | 35.9 | 78 | 7.8 | C10 | 123.4 d | Cu2+, Ni2+, Zn2+ | [56] |
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Park, J.E.; Jeong, G.S.; Lee, H.W.; Kim, S.K.; Kim, J.; Kim, H. Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library. Appl. Sci. 2021, 11, 5905. https://doi.org/10.3390/app11135905
Park JE, Jeong GS, Lee HW, Kim SK, Kim J, Kim H. Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library. Applied Sciences. 2021; 11(13):5905. https://doi.org/10.3390/app11135905
Chicago/Turabian StylePark, Jong Eun, Geum Seok Jeong, Hyun Woo Lee, Sung Kyum Kim, Jungho Kim, and Hoon Kim. 2021. "Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library" Applied Sciences 11, no. 13: 5905. https://doi.org/10.3390/app11135905
APA StylePark, J. E., Jeong, G. S., Lee, H. W., Kim, S. K., Kim, J., & Kim, H. (2021). Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library. Applied Sciences, 11(13), 5905. https://doi.org/10.3390/app11135905