Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine
Abstract
:1. Introduction
2. Results and Discussion
2.1. Purification and Identification of HSPHZZ
2.2. Gene Cloning and Sequence Analysis of HSPHZZ
2.3. Expression of Recombinant HSPHZZ in Escherichia coli BL21-Codon Plus (DE3)-RIL
2.4. Effect of Temperature and pH on Activity of the Recombinant HSPHZZ
2.5. Effect of Enzyme and Substrate Concentration on 2,5-DHP Production
2.6. Effect of Metal Ions, Organic Solvents, and Detergents on Enzymatic Activity
2.7. 2,5-DHP Production from HSP by HSPHZZ under Optimum Conditions
3. Experimental Section
3.1. Chemicals, Strains, and Plasmids
3.2. Purification of HSPHZZ
3.3. Enzymatic Activity Assay
3.4. N-Terminal Amino Acid Sequence of HSPHZZ
3.5. Gene Cloning and Construction of Expression Plasmid
3.6. Expression and Purification of Recombinant HSPHZZ
3.7. Catalytic Properties of Recombinant HSPHZZ
4. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Step | Total Protein (mg) | Total Activity (U) | Specific Activity (U/mg) | Fold | Yield (%) |
---|---|---|---|---|---|
Crude cell extract | 672.6 | 195.4 | 0.29 | 1 | 100 |
(NH4)2SO4 precipitation | 542.3 | 173.5 | 0.31 | 1.1 | 88.8 |
DEAE sepharose Phenyl sepharose | 75.2 22.3 | 100.3 54.5 | 1.3 2.4 | 4.6 8.4 | 51.3 27.8 |
Superdex-200 | 5.1 | 26.1 | 5.1 | 17.6 | 13.4 |
Substrate | kcat(s−1) | Km(mM) | kcat/Km(s−1 mM−1) |
---|---|---|---|
HSP | 2.1 ± 0.3 | 0.18 ± 0.04 | 11.7 ± 1.5 |
NADH | 1.3 ± 0.2 | 0.23 ± 0.03 | 5.7 ± 1.1 |
Metals or Inhibitors | Concentration | Relative Activity (%) |
---|---|---|
None | - | 100 |
Mg2+ | 5 mM | 86 ± 3 |
Zn2+ | 5 mM | 27 ± 2 |
Cu2+ | 5 mM | 23 ± 1 |
Ca2+ | 5 mM | 92 ± 2 |
Mn2+ | 5 mM | 87 ± 4 |
Fe2+ | 5 mM | 13 ± 2 |
Ni2+ | 5 mM | 81 ± 3 |
Co2+ | 5 mM | 31 ± 3 |
K+ | 5 mM | 101 ± 5 |
Na+ | 5 mM | 98 ± 2 |
EDTA | 0.5 mM 5 mM | 79 ± 2 85 ± 2 |
Metals or Inhibitors | Concentration (%) | Relative Activity (%) |
---|---|---|
None | — | 100 |
Methanol | 20 (v/v) | 61 ± 2 |
Acetone | 20 (v/v) | 59 ± 4 |
Chloroform | 20 (v/v) | 33 ± 2 |
Formaldehyde | 20 (v/v) | 23 ± 3 |
Toluene | 20 (v/v) | 28 ± 5 |
DMF | 20 (v/v) | 65 ± 2 |
DMSO | 20 (v/v) | 93 ± 5 |
Tween 20 | 1 (w/v) | 86 ± 5 |
Tween 40 | 1 (w/v) | 110 ± 4 |
Tween 80 | 1 (w/v) | 91 ± 1 |
Triton X-100 | 1 (w/v) | 48 ± 3 |
Span 20 | 1 (w/v) | 12 ± 4 |
Span 40 | 1 (w/v) | 17 ± 5 |
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Wei, T.; Zang, J.; Zheng, Y.; Tang, H.; Huang, S.; Mao, D. Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine. Catalysts 2017, 7, 257. https://doi.org/10.3390/catal7090257
Wei T, Zang J, Zheng Y, Tang H, Huang S, Mao D. Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine. Catalysts. 2017; 7(9):257. https://doi.org/10.3390/catal7090257
Chicago/Turabian StyleWei, Tao, Jie Zang, Yadong Zheng, Hongzhi Tang, Sheng Huang, and Duobin Mao. 2017. "Characterization of a Novel Nicotine Hydroxylase from Pseudomonas sp. ZZ-5 That Catalyzes the Conversion of 6-Hydroxy-3-Succinoylpyridine into 2,5-Dihydroxypyridine" Catalysts 7, no. 9: 257. https://doi.org/10.3390/catal7090257