Biotransformation of (–)-Isopulegol by Rhodococcus rhodochrous
"> Figure 1
<p>Structure 1: (–)-isopulegol.</p> "> Figure 2
<p>Thin sections of <span class="html-italic">R. rhodochrous</span> IEGM 1362 as viewed with TEM in cultures grown on nutrient agar (<b>a</b>–<b>c</b>) without (control) or (<b>d</b>–<b>f</b>) with (–)-isopulegol (0.025% <span class="html-italic">v</span>/<span class="html-italic">v</span>) and (<b>g</b>,<b>h</b>) in acute tests with the addition of (–)-isopulegol onto agar with already grown culture. Designations: C, capsular layer; CW, cell wall; CW Ls, cell wall layers; CM, cytoplasmic membrane; N, nucleoid; Pp, polyphosphate particles; L, lipid inclusions; MS, membrane-like structures; DC, destructed cells. Bar, 200 nm.</p> "> Figure 3
<p>Fluorescent microscopy (1000×) of <span class="html-italic">R</span>. <span class="html-italic">rhodochrous</span> IEGM 1362 cells stained with Nile Red: (<b>A</b>)—biotic control, (<b>B</b>)—in the presence of (–)-isopulegol, (<b>C</b>)—HNTs, (<b>D</b>)—(–)-isopulegol and HNTs. Arrows indicate lipid inclusions.</p> "> Figure 4
<p>Elemental composition of <span class="html-italic">R</span>. <span class="html-italic">rhodochrous</span> IEGM 1362 determined by mapping using X-ray microanalysis. Distribution of individual chemical elements is highlighted with color. Cell growth on: (<b>a</b>)—BTN agar, (<b>b</b>)—BTN agar plus (–)-isopulegol, (<b>c</b>)—BTN agar plus HNT, (<b>d</b>)—BTN agar plus (–)-isopulegol and HNTs, or (<b>e</b>)—in RS medium with (–)-isopulegol as the only carbon and energy source. The white arrow indicates a cell depleted in the set of elements and, apparently, inactive or dead.</p> "> Figure 5
<p>AFM and combined AFM-CLSM images of <span class="html-italic">R</span>. <span class="html-italic">rhodochrous</span> IEGM 1362 (biotic control <b>A</b>, <b>B</b>) in the presence of (–)-isopulegol (<b>C</b>,<b>D</b>), HNTs (<b>E</b>,<b>F</b>), and (–)-isopulegol and HNTs (<b>G</b>,<b>H</b>). Green fluorescence indicates living cells, red fluorescence indicates dead cells.</p> "> Figure 6
<p>Oxygen consumption rate (<b>A</b>) and growth dynamics (<b>B</b>) of R. rhodochrous IEGM 1362 in the presence of (‒)-isopulegol and HNTs (<span class="html-fig-inline" id="pharmaceuticals-15-00964-i001"> <img alt="Pharmaceuticals 15 00964 i001" src="/pharmaceuticals/pharmaceuticals-15-00964/article_deploy/html/images/pharmaceuticals-15-00964-i001.png"/></span>), (‒)-isopulegol (<span class="html-fig-inline" id="pharmaceuticals-15-00964-i002"> <img alt="Pharmaceuticals 15 00964 i002" src="/pharmaceuticals/pharmaceuticals-15-00964/article_deploy/html/images/pharmaceuticals-15-00964-i002.png"/></span>), and HNTs (<span class="html-fig-inline" id="pharmaceuticals-15-00964-i003"> <img alt="Pharmaceuticals 15 00964 i003" src="/pharmaceuticals/pharmaceuticals-15-00964/article_deploy/html/images/pharmaceuticals-15-00964-i003.png"/></span>). Biotic control (<span class="html-fig-inline" id="pharmaceuticals-15-00964-i004"> <img alt="Pharmaceuticals 15 00964 i004" src="/pharmaceuticals/pharmaceuticals-15-00964/article_deploy/html/images/pharmaceuticals-15-00964-i004.png"/></span>), abiotic control (<span class="html-fig-inline" id="pharmaceuticals-15-00964-i005"> <img alt="Pharmaceuticals 15 00964 i005" src="/pharmaceuticals/pharmaceuticals-15-00964/article_deploy/html/images/pharmaceuticals-15-00964-i005.png"/></span>).</p> "> Figure 7
<p>TLC of (–)-isopulegol (<b>a</b>) Rf 0.65 and its biotransformation products; (<b>b</b>) Rf 0.01 and 0.07 using <span class="html-italic">R</span>. <span class="html-italic">rhodochrous</span> IEGM 1362 cells.</p> "> Figure 8
<p>Chromatogram of the extract obtained during biotransformation of (–)-isopulegol (5 days) using <span class="html-italic">R. rhodochrous</span> IEGM 1362 in the presence of HNTs: <b>1</b>, (–)-isopulegol, <b>2</b>, metabolite <span class="html-italic">m/z</span> 170, <b>3</b>, metabolite <span class="html-italic">m/z</span> 184.</p> "> Figure 9
<p>Structures <b>2</b> and <b>2a</b>.</p> "> Figure 10
<p>Structures <b>3</b> and <b>3a</b>.</p> "> Figure 11
<p>Proposed pathway of (–)-isopulegol bioconversion by <span class="html-italic">R</span>. <span class="html-italic">rhodochrous</span> IEGM 1362.</p> "> Figure 12
<p>Structures <b>4</b>, <b>5</b> and <b>6</b>.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Screening for a Target Rhodococcus Strain and Evaluation of Its Response to (–)-Isopulegol and/or HNTs
2.2. Biotransformation of (–)-Isopulegol by R. rhodochrous IEGM 1362: The State and Activity of Cells
2.3. Identification of Products of (–)-Isopulegol Biotransformation
2.4. In Silico Analysis of (–)-Isopulegol and Its Derivatives
3. Materials and Methods
3.1. Microorganisms
3.2. Reagents and Nanomaterials
3.3. Cultivation Conditions
3.4. Phase-Contrast and Fluorescent Microscopy
3.5. Atomic Force and Confocal Laser Scanning Microscopy (AFM-CLSM)
3.6. Transmission Electron Microscopy (TEM)
3.7. Energy Dispersive X-ray Spectroscopy (EDX) with Elemental Mapping
3.8. Zeta Potential Measurement
3.9. Metabolic Activity Measurements
3.10. Extraction and Analysis of Residual (–)-Isopulegol and Its Derivatives
3.11. X-ray Data for Compounds 2 and 3
3.12. In Silico Analysis of (–)-Isopulegol and Its Derivatives
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AFM | atomic force microscopy |
CCDC | the Cambridge Crystallographic Data Center |
CLSM | confocal laser scanning microscopy |
EDX | energy dispersive X-ray spectrometry |
GC-MS | gas chromatography-mass spectrometry |
TEM | transmission electron microscopy |
TLC | thin layer chromatography |
HMBC | heteronuclear multiple bond correlation |
HNTs | halloysite nanotubes |
HR-MS | high resolution mass spectrometry |
NMR | nuclear magnetic resonance |
OD | optical density |
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Culture Conditions | Days | (–)-Isopulegol (m/z 154, 7.84 min) | Compound 2 (m/z 170, 10.66 min) | Compound 3 (m/z 184, 11.50 min) |
---|---|---|---|---|
Medium, 25 mL | ||||
(–)-Isopulegol | 2 | 15.2 | 84.8 | − |
3 | 45.7 | − | 54.3 | |
5 | 19.4 | − | 80.6 | |
(–)-Isopulegol and HNTs | 2 | 40.8 | 59.2 | − |
3 | − | 100 | − | |
5 | 12.0 | 72.2 | 15.7 | |
Medium, 100 mL | ||||
(–)-Isopulegol | 2 | − | − | 100 |
(–)-Isopulegol and HNTs | 2 | − | − | 100 |
Test Organisms (Index, Exposure) | Concentration, mg/L | ||
---|---|---|---|
(–)-Isopulegol | Compound 2 | Compound 3 | |
Water solubility at 25 °C | 308.6 | 345.2 | 6303.00 |
ECOSAR Class | Neutral Organics | Neutral Organics | Neutral Organics-acid |
Acute toxicity | |||
Fish (LD50, 96 h) | 7.39 | 72.75 | 1138.44 |
Daphnia (LD50, 48 h) | 4.76 | 42.53 | 654.68 |
Green algae (ED50, 96 h) | 5.99 | 35.74 | 513.99 |
Chronic toxicity | |||
Fish (ED50, 30 d) | 0.84 | 7.36 | 112.96 |
Daphnia (ED50, 21 d) | 0.66 | 4.49 | 66.15 |
Green algae (ED50, 16 d) | 2.08 | 9.98 | 138.47 |
Estimated Activity | (–)-Isopulegol | Compound 2 | Compound 3 | |||
---|---|---|---|---|---|---|
Pa | Pi | Pa | Pi | Pa | Pi | |
Carminative | 0.976 | 0.000 | 0.930 | 0.001 | 0.928 | 0.001 |
Anti–eczema | 0.929 | 0.004 | 0.918 | 0.004 | 0.908 | 0.004 |
Neuromuscular acetyl choline blocker | 0.751 | 0.003 | 0.677 | 0.005 | 0.682 | 0.005 |
Inhibitor of β-glucuronidase | – | – | 0.670 | 0.005 | 0.714 | 0.004 |
Immunosuppressive | 0.755 | 0.010 | 0.720 | 0.014 | 0.731 | 0.013 |
Stimulator of NFκB transcription factor | 0.747 | 0.003 | 0.730 | 0.004 | 0.730 | 0.004 |
Inhibitor of retinol dehydrogenase | 0.738 | 0.002 | 0.739 | 0.002 | 0.693 | 0.002 |
Respiratory analeptic | – | – | 0.568 | 0.026 | 0.686 | 0.016 |
Anti-inflammatory | 0.690 | 0.024 | 0.645 | 0.024 | 0.692 | 0.013 |
Antitumor | – | – | 0.550 | 0.057 | 0.482 | 0.002 |
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Ivshina, I.B.; Luchnikova, N.A.; Maltseva, P.Y.; Ilyina, I.V.; Volcho, K.P.; Gatilov, Y.V.; Korchagina, D.V.; Kostrikina, N.A.; Sorokin, V.V.; Mulyukin, A.L.; et al. Biotransformation of (–)-Isopulegol by Rhodococcus rhodochrous. Pharmaceuticals 2022, 15, 964. https://doi.org/10.3390/ph15080964
Ivshina IB, Luchnikova NA, Maltseva PY, Ilyina IV, Volcho KP, Gatilov YV, Korchagina DV, Kostrikina NA, Sorokin VV, Mulyukin AL, et al. Biotransformation of (–)-Isopulegol by Rhodococcus rhodochrous. Pharmaceuticals. 2022; 15(8):964. https://doi.org/10.3390/ph15080964
Chicago/Turabian StyleIvshina, Irina B., Natalia A. Luchnikova, Polina Yu. Maltseva, Irina V. Ilyina, Konstantin P. Volcho, Yurii V. Gatilov, Dina V. Korchagina, Nadezhda A. Kostrikina, Vladimir V. Sorokin, Andrey L. Mulyukin, and et al. 2022. "Biotransformation of (–)-Isopulegol by Rhodococcus rhodochrous" Pharmaceuticals 15, no. 8: 964. https://doi.org/10.3390/ph15080964