Lignin Monomers: Oxidation and Degradation - Revision history

Bes at 13:38, 2 August 2020

2020-08-02T13:38:00Z

Ztaylor: /* Conclusion */

2020-05-15T04:16:13Z

Conclusion

Ztaylor: /* Discussion */

2020-05-15T04:14:16Z

Discussion

Ztaylor: /* Discussion */

2020-05-15T04:14:03Z

Discussion

Ztaylor: /* EPR Analysis */

2020-05-15T04:13:34Z

EPR Analysis

Ztaylor: /* EPR Analysis with Immobilized Enzyme */

2020-05-15T04:13:01Z

EPR Analysis with Immobilized Enzyme

Ztaylor: /* Discussion */

2020-05-15T04:10:09Z

Discussion

Ztaylor: /* Comparison of Ferulic Acid and Coniferyl Alcohol Using WebMO Calculations & WINSIM Simulation */

2020-05-15T04:08:17Z

Comparison of Ferulic Acid and Coniferyl Alcohol Using WebMO Calculations & WINSIM Simulation

Ztaylor: /* EPR Analysis */

2020-05-15T04:07:12Z

EPR Analysis

Ztaylor: /* EPR Analysis */

2020-05-15T04:05:24Z

EPR Analysis

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		+	'''''The following document was written by Zelinda Taylor as partial fulfillment of her graduation requirements for a Bachelors Degree of Biochemistry, Monmouth College, spring 2020.'''''
	==Abstract==		==Abstract==

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−	Little progress was made on the investigation into the pathway of formation of ~~lignin~~. Two different approaches were used, but no conclusive results were developed for either of these. The degradation method presented the most promising results and literature is relatively abundant on the topic. More literature review should be done to determine a degradation method that can be completed and then analyzed on the HPLC. A successful method will assist with determining how lignan forms. This pathway can then be compared to the pathway of formation of lignin. Other lignans could also be researched to identify oxidation products of the monomers that could be dimers.	+	Little progress was made on the investigation into the pathway of formation of lignan. Two different approaches were used, but no conclusive results were developed for either of these. The degradation method presented the most promising results and literature is relatively abundant on the topic. More literature review should be done to determine a degradation method that can be completed and then analyzed on the HPLC. A successful method will assist with determining how lignan forms. This pathway can then be compared to the pathway of formation of lignin. Other lignans could also be researched to identify oxidation products of the monomers that could be dimers.

	==Future Directions==		==Future Directions==

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−	Flat cell reactions were completed using HRP<sub>i</sub> and H<sub>2</sub>O<sub>2</sub> and analyzed on the HPLC to examine if oxidation products were being formed before analyzing the reactions using ~~EPR~~. Products were produced meaning the HRP<sub>i</sub> was functioning and the reaction setup of the flat cell was adequate. Using the ESR for analysis, the coupling constants of the assumed radicals formed during the oxidation reaction were of interest. These constants could be determined using computational chemistry software, as well as simulations of the experimental data collected. Completing both of these analyses and comparing them showed radials were more likely to be formed at the C-5 and C-β positions than other positions on the molecule. These findings agreed with previous data collected by ''Russell, Forrester, Chesson, and Burkitt.'' The coupling constants of the assumed radials formed were of interest because they could be used as a way to predict how oxidative coupling occurs. The larger structure of lignin could be formed vs the dimers, known as lignan. By determining the coupling constants, the oxidative coupling process can be further studied to determine which structures are likely to couple together, and how they couple.	+	Flat cell reactions were completed using HRP<sub>i</sub> and H<sub>2</sub>O<sub>2</sub> and analyzed on the HPLC to examine if oxidation products were being formed before analyzing the reactions using ESR. Products were produced meaning the HRP<sub>i</sub> was functioning and the reaction setup of the flat cell was adequate. Using the ESR for analysis, the coupling constants of the assumed radicals formed during the oxidation reaction were of interest. These constants could be determined using computational chemistry software, as well as simulations of the experimental data collected. Completing both of these analyses and comparing them showed radials were more likely to be formed at the C-5 and C-β positions than other positions on the molecule. These findings agreed with previous data collected by ''Russell, Forrester, Chesson, and Burkitt.'' The coupling constants of the assumed radials formed were of interest because they could be used as a way to predict how oxidative coupling occurs. The larger structure of lignin could be formed vs the dimers, known as lignan. By determining the coupling constants, the oxidative coupling process can be further studied to determine which structures are likely to couple together, and how they couple.

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−	Flat cell reactions were completed using HRP<sub>i</sub> and H<sub>2</sub>O<sub>2</sub> and analyzed on the HPLC to examine if oxidation products were being formed before analyzing the reactions using EPR. Products were produced meaning the HRP<sub>i</sub> was functioning and the reaction setup of the flat cell was adequate. Using the ~~EPR~~ for analysis, the coupling constants of the assumed radicals formed during the oxidation reaction were of interest. These constants could be determined using computational chemistry software, as well as simulations of the experimental data collected. Completing both of these analyses and comparing them showed radials were more likely to be formed at the C-5 and C-β positions than other positions on the molecule. These findings agreed with previous data collected by ''Russell, Forrester, Chesson, and Burkitt.'' The coupling constants of the assumed radials formed were of interest because they could be used as a way to predict how oxidative coupling occurs. The larger structure of lignin could be formed vs the dimers, known as lignan. By determining the coupling constants, the oxidative coupling process can be further studied to determine which structures are likely to couple together, and how they couple.	+	Flat cell reactions were completed using HRP<sub>i</sub> and H<sub>2</sub>O<sub>2</sub> and analyzed on the HPLC to examine if oxidation products were being formed before analyzing the reactions using EPR. Products were produced meaning the HRP<sub>i</sub> was functioning and the reaction setup of the flat cell was adequate. Using the ESR for analysis, the coupling constants of the assumed radicals formed during the oxidation reaction were of interest. These constants could be determined using computational chemistry software, as well as simulations of the experimental data collected. Completing both of these analyses and comparing them showed radials were more likely to be formed at the C-5 and C-β positions than other positions on the molecule. These findings agreed with previous data collected by ''Russell, Forrester, Chesson, and Burkitt.'' The coupling constants of the assumed radials formed were of interest because they could be used as a way to predict how oxidative coupling occurs. The larger structure of lignin could be formed vs the dimers, known as lignan. By determining the coupling constants, the oxidative coupling process can be further studied to determine which structures are likely to couple together, and how they couple.

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 To evaluate the use of the immobilized enzyme in a flat cell, samples of the waste were collected to ensure the immobilized enzyme was functioning in the flat cell. To do this, a flat cell was loaded with steel wool, sand, and agarose beads with HRPi. MOPS Buffer was then ran over the beads in the flat cell to wash them and ensure they were not dry. A substrate of 1 mM ferulic acid and 1 mM hydrogen peroxide in 50/50 dioxane/pH 5 buffer was then ran over the immobilized enzyme in the flat cell. The flow of the substrate was controlled using a syringe pump at 0.25 mL/min, 0.5 mL/min, 1 mL/min, and 2 mL/min. Samples of waste were then collected at each flow rate and analyzed on the HPLC. The results of ferulic acid are shown in ''figure 7.'' The same procedure was used for a substrate of 1 mM p-coumaric acid and 1 mM hydrogen peroxide in 50/50 dioxane/pH 5 buffer and is shown in ''figure 8''.
-====EPR Analysis====
+====ESR Analysis====
 [[File:Ligin Monomer radicals.png|300px|thumb|center|Figure 9: ESR data from HRP oxidation of lignin monomers]]
-''Figure 9'' shows previously collected EPR data of the oxidation of lignin monomers. In ''figure 10'', ''figure 11'', ''figure 12'' and ''figure 13'', preliminary ESR data is shown. To complete preliminary ESR data collection using ferulic acid, a substrate of 10 mM ferulic acid in 50/50 dioxane/pH 5 buffer was made. The flat cell was then loaded with steel wool, sand, and immobilized enzyme. Buffer was ran over the immobilized enzyme for about an hour. The flat cell was then mounted in the ESR. Hydrogen peroxide was added to the substrate directly before flow was began so its concentration in solution was 10 mM. The substrate was then flowed through the flat cell in the ESR at a flow rate of 1 mL/min and a signal was collected. The flow of the substrate was then increased to 2 mL/min and a signal was collected. The flow of the substrate was then stopped, and no signal was apparent. This was done to ensure the signal being produced was a result of the oxidation of the monomers. If this was true, stopping the flow would stop the signal. The flow was then re-started with a flow rate of 2 mL/min, and the signal returned.
+''Figure 9'' shows previously collected ESR data of the oxidation of lignin monomers. In ''figure 10'', ''figure 11'', ''figure 12'' and ''figure 13'', preliminary ESR data is shown. To complete preliminary ESR data collection using ferulic acid, a substrate of 10 mM ferulic acid in 50/50 dioxane/pH 5 buffer was made. The flat cell was then loaded with steel wool, sand, and immobilized enzyme. Buffer was ran over the immobilized enzyme for about an hour. The flat cell was then mounted in the ESR. Hydrogen peroxide was added to the substrate directly before flow was began so its concentration in solution was 10 mM. The substrate was then flowed through the flat cell in the ESR at a flow rate of 1 mL/min and a signal was collected. The flow of the substrate was then increased to 2 mL/min and a signal was collected. The flow of the substrate was then stopped, and no signal was apparent. This was done to ensure the signal being produced was a result of the oxidation of the monomers. If this was true, stopping the flow would stop the signal. The flow was then re-started with a flow rate of 2 mL/min, and the signal returned.

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 A flat cell was loaded with glass wool, sand, and immobilized enzyme. Glass wool was lightly packed into the bottom neck of the flat cell. A small amount of sand was then mixed with nanopure water and added into the flat cell about 2 mm above the glass wool using a plastic 1 mL syringe. About 50 μL of immobilized enzyme is added on top of the glass wool and sand to fill most of the cavity of the flat cell. The pH 5 buffer was ran through the flat cell for about 10 minutes. A 1 mM ferulic acid, 1 mM hydrogen peroxide solution in 50/50 dioxane/pH 5 buffer was made. This solution was put in a 60 mL plastic syringe and flow was controlled using a syringe pump at 0.25 mL/minute, 0.5 mL/minute, 1 mL/minute, and 2 mL/minute. Flow was collected for each rate and analyzed on the HPLC. The same procedure was completed for p-coumaric acid.
-===EPR Analysis with Immobilized Enzyme===
+===ESR Analysis with Immobilized Enzyme===
-A flat cell was loaded with glass wool, sand, and immobilized enzyme using the procedure outlined above. A 10 mM ferulic acid solution in 50/50 dioxane/pH 7.4 buffer was made. Hydrogen peroxide was added to a concentration of 10 mM before EPR analysis. The solution was placed in a 60 mL plastic syringe and flow was controlled. Flow was analyzed at rates of 0.5 mL/minute, 1 mL/minute, and 2 mL/minute. The parameters for the program were modulation amplitude 0.5 G, sweep width 40.0 G, and center field at 3492.0 G.
+A flat cell was loaded with glass wool, sand, and immobilized enzyme using the procedure outlined above. A 10 mM ferulic acid solution in 50/50 dioxane/pH 7.4 buffer was made. Hydrogen peroxide was added to a concentration of 10 mM before ESR analysis. The solution was placed in a 60 mL plastic syringe and flow was controlled. Flow was analyzed at rates of 0.5 mL/minute, 1 mL/minute, and 2 mL/minute. The parameters for the program were modulation amplitude 0.5 G, sweep width 40.0 G, and center field at 3492.0 G.
 ===Lignan Beaker Reactions===

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−	With the use of HRP and H<sub>2</sub>O<sub>2</sub>, lignan monomers were able to be oxidized and analyzed on the HPLC using an acetonitrile and water gradient. Concentration dependent reactions were made so the substrate decreased linearly as the concentration of H<sub>2</sub>O<sub>2</sub> within the reaction was increased. While the decrease of substrate was important, the products produced for each reaction were of interest. As the substrate was decreasing, or being consumed, the amount of products detected should increase. This was the case with the ferulic acid as well as the p-coumaric acid. For sinapic acid, this did not occur. The substrate was not consumed and products were not formed. Throughout each reaction, as the concentration of H<sub>2</sub>O<sub>2</sub> was increased, the same, prominent substrate peak was present. The lack of products could be due to the double-methoxy substitutions on the phenol. Ferulic acid only posses one, and p-coumaric acid possess none. It is possible the two methoxy groups of sinapic acid restrict oxidation, therefore forming no products. These same results were observed when using both HRP and immobilized HRP (HRP<sub>i</sub>) in beaker reactions.	+	With the use of HRP and H<sub>2</sub>O<sub>2</sub>, lignan monomers were able to be oxidized and analyzed on the HPLC using an acetonitrile and water gradient. Concentration dependent reactions were made so the substrate decreased linearly as the concentration of H<sub>2</sub>O<sub>2</sub> within the reaction was increased. While the decrease of substrate was important, the products produced for each reaction were of interest. As the substrate was decreasing, or being consumed, the amount of products detected should increase. This was the case with the ferulic acid as well as the p-coumaric acid. For sinapic acid, this did not occur. The substrate was not consumed and products were not formed. Throughout each reaction, as the concentration of H<sub>2</sub>O<sub>2</sub> was increased, the same, prominent substrate peak was present. The lack of products could be due to the double-methoxy substitutions on the phenol. Ferulic acid only posses one, and p-coumaric acid possess none. It is possible the two methoxy groups of sinapic acid restrict oxidation, therefore forming no products. These same results were observed when using both HRP and immobilized HRP (HRP<sub>i</sub>) in beaker reactions. Once the substrate was limited to pH 5 buffer only, some product peaks formed opposed to none with the substrate in pH 5 buffer and dioxane.

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−	The above table shows the comparison of radical forms of coniferyl alcohol and ferulic acid. The table shows the experimental simulation values, previous collected values, and the WebMO calculation values. These values collected at this institution are on the figures below, the experimental simulation values are in bold and the WebMO 18 calculation values are in standard text.	+	The above table shows the comparison of radical forms of coniferyl alcohol and ferulic acid. The table shows the experimental simulation values, previous collected values, and the WebMO 18 calculation values. These values collected at this institution are on the figures below, the experimental simulation values are in bold and the WebMO 18 calculation values are in standard text.

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−	''Figure 9'' shows previously collected EPR data of the oxidation of lignin monomers. In ''figure 10'', ''figure 11'', ''figure 12'' and ''figure 13'', preliminary ESR data is shown. To complete preliminary ESR data collection using ferulic acid, a substrate of 1 mM ferulic acid in 50/50 dioxane/pH 5 buffer was made. The flat cell was then loaded with steel wool, sand, and immobilized enzyme. Buffer was ran over the immobilized enzyme for about an hour. The flat cell was then mounted in the ESR. Hydrogen peroxide was added to the substrate directly before flow was began so its concentration in solution was 1 mM. The substrate was then flowed through the flat cell in the ESR at a flow rate of 1 mL/min and a signal was collected. The flow of the substrate was then increased to 2 mL/min and a signal was collected. The flow of the substrate was then stopped, and no signal was apparent. This was done to ensure the signal being produced was a result of the oxidation of the monomers. If this was true, stopping the flow would stop the signal. The flow was then re-started with a flow rate of 2 mL/min, and the signal returned.	+	''Figure 9'' shows previously collected EPR data of the oxidation of lignin monomers. In ''figure 10'', ''figure 11'', ''figure 12'' and ''figure 13'', preliminary ESR data is shown. To complete preliminary ESR data collection using ferulic acid, a substrate of 10 mM ferulic acid in 50/50 dioxane/pH 5 buffer was made. The flat cell was then loaded with steel wool, sand, and immobilized enzyme. Buffer was ran over the immobilized enzyme for about an hour. The flat cell was then mounted in the ESR. Hydrogen peroxide was added to the substrate directly before flow was began so its concentration in solution was 10 mM. The substrate was then flowed through the flat cell in the ESR at a flow rate of 1 mL/min and a signal was collected. The flow of the substrate was then increased to 2 mL/min and a signal was collected. The flow of the substrate was then stopped, and no signal was apparent. This was done to ensure the signal being produced was a result of the oxidation of the monomers. If this was true, stopping the flow would stop the signal. The flow was then re-started with a flow rate of 2 mL/min, and the signal returned.

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−	''Figure 9'' shows previously collected EPR data of the oxidation of lignin monomers. In ''figure 10'', ''figure 11'', ''figure 12'' and ''figure 13'', preliminary ESR data is shown To complete preliminary ESR data collection using ferulic acid, a substrate of 1 mM ferulic acid in 50/50 dioxane/pH 5 buffer was made. The flat cell was then loaded with steel wool, sand, and immobilized enzyme. Buffer was ran over the immobilized enzyme for about an hour. The flat cell was then mounted in the ESR. Hydrogen peroxide was added to the substrate directly before flow was began so its concentration in solution was 1 mM. The substrate was then flowed through the flat cell in the ESR at a flow rate of 1 mL/min and a signal was collected. The flow of the substrate was then increased to 2 mL/min and a signal was collected. The flow of the substrate was then stopped, and no signal was apparent. This was done to ensure the signal being produced was a result of the oxidation of the monomers. If this was true, stopping the flow would stop the signal. The flow was then re-started with a flow rate of 2 mL/min, and the signal returned.	+	''Figure 9'' shows previously collected EPR data of the oxidation of lignin monomers. In ''figure 10'', ''figure 11'', ''figure 12'' and ''figure 13'', preliminary ESR data is shown. To complete preliminary ESR data collection using ferulic acid, a substrate of 1 mM ferulic acid in 50/50 dioxane/pH 5 buffer was made. The flat cell was then loaded with steel wool, sand, and immobilized enzyme. Buffer was ran over the immobilized enzyme for about an hour. The flat cell was then mounted in the ESR. Hydrogen peroxide was added to the substrate directly before flow was began so its concentration in solution was 1 mM. The substrate was then flowed through the flat cell in the ESR at a flow rate of 1 mL/min and a signal was collected. The flow of the substrate was then increased to 2 mL/min and a signal was collected. The flow of the substrate was then stopped, and no signal was apparent. This was done to ensure the signal being produced was a result of the oxidation of the monomers. If this was true, stopping the flow would stop the signal. The flow was then re-started with a flow rate of 2 mL/min, and the signal returned.