Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library
Abstract
:1. Introduction
2. Results and Discussion
2.1. Construction of Metagenomic Library and Isolation of Agarase Gene
2.2. Sequence Analysis
2.3. Expression and Purification of Recombination Agarase
2.4. Effects of pH and Temperature on the Activity of Recombination Agarase
2.5. Effects of Various Metal Ions and Reagents on the Activity of Recombination Agarase
2.6. Analysis of the Hydrolysis Pattern and Products of Recombination Agarase
3. Materials and Methods
3.1. Strains, Plasmids, and Culture Conditions
3.2. Construction of Metagenomic Library
3.3. Library Screening and AgaML Gene Isolation
3.4. Sequence Analysis and Classification of AgaML
3.5. Cloning and Expression of AgaML
3.6. Purification of Recombinant Agarase
3.7. Agarase Activity Assay
3.8. Effects of pH and Temperature on Recombinant Agarase Activity
3.9. Effects of Various Metal Ions and Reagents on Recombinant Agarase Activity
3.10. Hydrolysis Products Analysis of Recombinant Agarase
3.11. Nucleotide Sequence Accession Number
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Reagents | Concentration (mM) | Relative Activity (%) a |
---|---|---|
None | - | 100.0 ± 4.2 b |
Na+ | 100 | 103 ± 3.4 |
K+ | 100 | 104 ± 3.5 |
NH4+ | 100 | 69 ± 5.6 |
Mn2+ | 1 | 115 ± 4.3 |
Mg2+ | 1 | 87 ± 4.6 |
Fe3+ | 1 | 98 ± 3.2 |
Zn2+ | 1 | 74 ± 6.2 |
Ca2+ | 1 | 108 ± 6.4 |
Cu2+ | 1 | 75 ± 5.1 |
Ba2+ | 1 | 113 ± 4.2 |
Co2+ | 1 | 103 ± 3.6 |
Cd2+ | 1 | 85 ± 4.3 |
EDTA | 10 | 17 ± 5.7 |
SDS | 10 | 73 ± 5.3 |
Family | Protein | Strain | Products | References |
---|---|---|---|---|
GH16 | AgaML | Metagenomic library | NA4, NA6 | This study |
AgaA | Pseudoalteromonas sp. CY24 | NA2, NA4, NA6 | [38] | |
AgaG1 | Alteromonas sp. GNUM1 | NA2, NA4 | [39] | |
AgaA | Agarivorans sp. LQ48 | NA4, NA6 | [27] | |
AgaYT | Flammeovirga yaeyamensis | NA2, NA4 | [40] | |
GH50 | RagaA11 | Agarivorans sp. JAMB-A11 | NA2 | [41] |
Unnamed | Agarivorans sp. JA-1 | NA2, NA4 | [11] | |
AgWH50A | Agarivorans gilus WH0801 | NA4 | [42] | |
GH86 | AgaP4383 | Flammeovirga pacifica WPAGA1 | NA4, NA6 | [37] |
AgaO | Microbulbifer sp. JAMB-A94 | NA6 | [43] | |
AgaA | Cellvibrio sp. OA-2007 | NA2, NA4 | [44] | |
GH118 | AgaXa | Catenovulum sp. X3 | NA6, NA8, NA10, NA12 | [45] |
Agarase-a | Agarivorans albus OAY02 | NA2, NA4, NA6 | [46] | |
AgaB | Pseudoalteromonas sp. CY24 | NA8, NA10 | [47] |
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Mai, Z.; Su, H.; Zhang, S. Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library. Int. J. Mol. Sci. 2016, 17, 1360. https://doi.org/10.3390/ijms17081360
Mai Z, Su H, Zhang S. Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library. International Journal of Molecular Sciences. 2016; 17(8):1360. https://doi.org/10.3390/ijms17081360
Chicago/Turabian StyleMai, Zhimao, Hongfei Su, and Si Zhang. 2016. "Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library" International Journal of Molecular Sciences 17, no. 8: 1360. https://doi.org/10.3390/ijms17081360