Enzymatic Mechanism of Oxalate Production in the TCA  and Glyoxylate Pathways using Antrodia radiculosa                   ...
Objective   Gain insight into the    biosynthesis of    oxalate in order to    understand the    mechanism utilized    by...
Background   Copper-tolerant fungi produce and accumulate    HIGH levels of oxalate   Oxalate biosynthesis has been link...
4
Methods                                   Antrodia radiculosa   Treatment:                          Isolates       Untre...
Methods   Enzyme Analysis                           Enzyme            Wavelength       Prepping the sample          Fuma...
7RESULTS – OXALATE                                         Oxalate Concentration                        1.4               ...
8RESULTS – ISOCITRATE LYASE ACTIVITY                                             ISOCITRATE LYASE ACTIVITY: TFFH 294      ...
9RESULTS – GLYOXYLATE DEHYDROGENASE ACTIVITY                                         GLYOXYLATE DEHYDROGENASE ACTIVITY: TF...
10RESULTS – FUMARASE ACTIVITY                                              FUMARASE ACTIVITY: TFFH 294                    ...
11RESULTS – MALATE SYNTHASE ACTIVITY                                         MALATE SYNTHASE ACTIVITY: TFFH 294           ...
Discussion - Oxalate   In general, oxalate concentration was highest    at Week 6 for all isolates treated with copper   ...
Discussion – Enzymatic Analysis   Isocitrate Lyase and Glyoxylate    Dehydrogenase activity     Specific   Activity and ...
Conclusion   Theoretically, oxalate production relies on    Isocitrate Lyase and Glyoxylate Dehydrogenase                ...
1515
Future Objectives   Evaluate Protein Concentration   Gene expression analysis   Copper Influence   Citrate            ...
Special Thanks         Forest Products Laboratory - Madison, WI             Carol Clausen             Rick Green      ...
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Session 28 ic2011 jenkins

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Session 28 ic2011 jenkins

  1. 1. Enzymatic Mechanism of Oxalate Production in the TCA and Glyoxylate Pathways using Antrodia radiculosa Katie M. Jenkins Department of Forest Products, Mississippi State University USDA FS, Forest Products Laboratory, Madison, WI
  2. 2. Objective Gain insight into the biosynthesis of oxalate in order to understand the mechanism utilized by varying isolates of Antrodia radiculosa 2
  3. 3. Background Copper-tolerant fungi produce and accumulate HIGH levels of oxalate Oxalate biosynthesis has been linked to the Glyoxylate and Tricarboxylic Acid (TCA) Pathways  Oxalateproduction relies on twelve specific enzymes  Two major oxalate producing enzymes:  GlyoxylateDehydrogenase  Oxaloacetase Munir, et al., 2001. PNAS 98(20): 11126-11130. 3
  4. 4. 4
  5. 5. Methods Antrodia radiculosa Treatment: Isolates  Untreated (Controls) FP-90848-T  1.2% Copper Citrate L-11659-SP Timeline: TFFH 294  2 weeks L-9414-SP  4 weeks  6 weeks  8 weeks Oxalate Analysis:  Trinity Biotech Reagents  560nm 5
  6. 6. Methods Enzyme Analysis Enzyme Wavelength  Prepping the sample Fumarase 240nm  Rasped into saw dust Malate Synthase 412nm  ~ 0.2g Oxaloacetase 255nm  Frozen in liquid N2 Isocitrate Lyase 324nm  Stored at -80°C Glyoxylate Dehydrogenase 550nm  Determining Activity  Spectrophotometrically  Biotech Epoch  96-well plates  UV  VIS 6
  7. 7. 7RESULTS – OXALATE Oxalate Concentration 1.4 1.2 1.0Concentration (mg/mL) L-11659-SP Copper Citrate 0.8 L-11659-SP Untreated FP-90848-T Copper Citrate FP-90848-T Untreated L-9414-SP Copper Citrate 0.6 L-9414-SP Untreated TFFH 294 Copper Citrate TFFH 294 Untreated 0.4 0.2 0.0 2 Weeks 4 Weeks 6 Weeks 8 Weeks
  8. 8. 8RESULTS – ISOCITRATE LYASE ACTIVITY ISOCITRATE LYASE ACTIVITY: TFFH 294 1.1 1.0 0.9 0.8Change in Absorbance/Minute 0.7 Copper Citrate Week 2 0.6 Untreated Week 2 Copper Citrate Week 4 0.5 Untreated Week 4 Copper Citrate Week 6 Untreated Week 6 0.4 Copper Citrate Week 8 Untreated Week 8 0.3 0.2 0.1 0.0 0 10 20 30 40 50 60 Enzyme Filtrate (ul)
  9. 9. 9RESULTS – GLYOXYLATE DEHYDROGENASE ACTIVITY GLYOXYLATE DEHYDROGENASE ACTIVITY: TFFH 294 0.35 0.30Change in Absorbance/Minute 0.25 Copper Citrate Week 2 0.20 Untreated Week 2 Copper Citrate Week 4 Untreated Week 4 0.15 Copper Citrate Week 6 Untreated Week 6 Copper Citrate Week 8 Untreated Week 8 0.10 0.05 0.00 0 10 20 30 40 50 60 Enzyme Filtrate (ul)
  10. 10. 10RESULTS – FUMARASE ACTIVITY FUMARASE ACTIVITY: TFFH 294 0.5 0.4Change in Absorbance/Minute 0.3 Copper Citrate Week 2 Untreated Week 2 Copper Citrate Week 4 0.2 Untreated Week 4 Copper Citrate Week 6 Untreated Week 6 Copper Citrate Week 8 Untreated Week 8 0.1 0 0 10 20 30 40 50 60 -0.1 Enzyme Filtrate (ul)
  11. 11. 11RESULTS – MALATE SYNTHASE ACTIVITY MALATE SYNTHASE ACTIVITY: TFFH 294 0.16 0.14 0.12Change in Absorbance/Minute 0.10 Copper Citrate Week 2 Untreated Week 2 0.08 Copper Citrate Week 4 Untreated Week 4 Copper Citrate Week 6 0.06 Untreated Week 6 Copper Citrate Week 8 Untreated Week 8 0.04 0.02 0.00 0 10 20 30 40 50 60 Enzyme Filtrate (ul)
  12. 12. Discussion - Oxalate In general, oxalate concentration was highest at Week 6 for all isolates treated with copper citrate.  Exceptions:  L-11659-SP – Week 2  FP-90848-T – Week 8 No observable variation in oxalate production between untreated and copper citrate treated blocks.  Week 8 12
  13. 13. Discussion – Enzymatic Analysis Isocitrate Lyase and Glyoxylate Dehydrogenase activity  Specific Activity and Total Protein Fumarase and Oxaloacetase show NO activity Malate Synthase showing odd behavior when treated with copper citrate  Untreated samples have NO activity  Possible substrate inhibition 13
  14. 14. Conclusion Theoretically, oxalate production relies on Isocitrate Lyase and Glyoxylate Dehydrogenase 14
  15. 15. 1515
  16. 16. Future Objectives Evaluate Protein Concentration Gene expression analysis Copper Influence Citrate 16
  17. 17. Special Thanks  Forest Products Laboratory - Madison, WI  Carol Clausen  Rick Green  Mississippi State University  Dr. Susan Diehl  Dr. Ken Willeford  Juliet Tang 17

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