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Sasithorn Kongruang

. Dissertation Topics. Quantitative Analysis of Cellulose Reducing Ends Colorimetric assaysRadioisotope assaysBorohydride Reactivity of Cellulose Reducing Ends Time course analysisDetecting Enzyme Activity: A Case Study of Polygalacturonase. Cellulose. Cellobiose (?-1,4-glycosidic linkage).

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Sasithorn Kongruang

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    2. Hi Committee members, My name is Sasithorn Kongruang. Today I would like to present my oral preliminary exam in four topics Hi Committee members, My name is Sasithorn Kongruang. Today I would like to present my oral preliminary exam in four topics

    3. Cellulose

    8. How to express substrate concentration ? Enzyme accessible surface area (cellulose surface area) Solute Exclusion Techniques the area corresponding to the cellulose pores can be evaluated, but the external surface (nonporous area) is considered negligible. The cellulose pore volume accessible to probes of different sizes-glucose, cellobiose, polyethylene glycol. The solute exclusion technique. This technique seems to better represent the amount of substrate actually available to enzymes. The area corresponding to the cellulose pores can be evaluated, but the external surface (nonporous area) is considered negligible. The cellulose pore volume accessible to probes of different sizes-glucose, cellobiose and polyethylene glycol.The solute exclusion technique. This technique seems to better represent the amount of substrate actually available to enzymes. The area corresponding to the cellulose pores can be evaluated, but the external surface (nonporous area) is considered negligible. The cellulose pore volume accessible to probes of different sizes-glucose, cellobiose and polyethylene glycol.

    9. Questions How many reducing end per unit mass substrate (MCC, AMCC, PSC, BMCC, ABMCC and FP) ? What percentage of total reducing end are solvent accessible ? So, the objective of this sectioon of my reserach is How many reducing ends per unit mass substrate ? What percentage of total reducing end are solvent accessible? What percentage of total reducing end are enzyme accessible? So, the objective of this sectioon of my reserach is How many reducing ends per unit mass substrate ? What percentage of total reducing end are solvent accessible? What percentage of total reducing end are enzyme accessible?

    10. Determination of insoluble reducing chain ends Methods used: Dinitrosalicylic acid:DNS assay (Irwin et al., 1993) Cu2+/bicinchoninic acid:BCA assay (Johnston et al., 1998) Cu2+/arsenomolybdate :Nelson assay (Ståhlberg et al., 1993 ) p-hydroxybenzoic acid hydrazine:PAHBAH assay (Boraston et al., 2001) For the determination of insoluble reducing chains ends: There are 4 methods used: 1) Dinitrosalicylic acid : DNS assay 2) Cu/bicinchoninic acid 3)Cu/arsenomolybdate 4) parahydroxybenzoic acid hydrazineFor the determination of insoluble reducing chains ends: There are 4 methods used: 1) Dinitrosalicylic acid : DNS assay 2) Cu/bicinchoninic acid 3)Cu/arsenomolybdate 4) parahydroxybenzoic acid hydrazine

    11. Reducing Sugar Assay BCA assay: Cu2+ is reduced to Cu+1, which is then specially chelated to form a colored complex. Reducing sugar + Cu2+ Oxidized sugar + Cu+1 DNS assay: the title compound are reduced, resulting in a shift in their visible absorption spectrum. Reducing sugar + Oxidized sugar + The chemical reaction behind the reducing sugar assay is For BCA Cu2+ is reduced to Cu+1, which is then chelated to form a colored complex For the DNS, the title compound are reduced, resulting in a shift in their visible absorption spectrumThe chemical reaction behind the reducing sugar assay is For BCA Cu2+ is reduced to Cu+1, which is then chelated to form a colored complex For the DNS, the title compound are reduced, resulting in a shift in their visible absorption spectrum

    12. The Solvent Accessible Reducing Ends Among the microcrystalline celluloses, they had the relatively same number of total of reducing ends, where approximately 66 % of those reducing ends are located at the surface. For the bacterial microcrystalline cellulose form (BMCC and AMCC), there are more reducing ends located at the surface of their structures. The 79% reducing ends exposed whereas the rest is still hidden in the crystalline structure.Among the microcrystalline celluloses, they had the relatively same number of total of reducing ends, where approximately 66 % of those reducing ends are located at the surface. For the bacterial microcrystalline cellulose form (BMCC and AMCC), there are more reducing ends located at the surface of their structures. The 79% reducing ends exposed whereas the rest is still hidden in the crystalline structure.

    13. Reduction/incorporation of Aldose to Alditol with NaBH4 / NaB3H4

    14. Time Course of the reduction of celluloses with NaBH4 As expected, the reducing ends that are present in the solution would have more freedom to react with sodium borohydride. Therefore, the rate of reduction would be faster than the reducing ends, which were attach on the cellulose surface. As expected, the reducing ends that are present in the solution would have more freedom to react with sodium borohydride. Therefore, the rate of reduction would be faster than the reducing ends, which were attach on the cellulose surface.

    15. The Solvent Accessible Reducing Ends Among the microcrystalline celluloses, they had the relatively same number of total of reducing ends, where approximately 66 % of those reducing ends are located at the surface. For the bacterial microcrystalline cellulose form (BMCC and AMCC), there are more reducing ends located at the surface of their structures. The 79% reducing ends exposed whereas the rest is still hidden in the crystalline structure.Among the microcrystalline celluloses, they had the relatively same number of total of reducing ends, where approximately 66 % of those reducing ends are located at the surface. For the bacterial microcrystalline cellulose form (BMCC and AMCC), there are more reducing ends located at the surface of their structures. The 79% reducing ends exposed whereas the rest is still hidden in the crystalline structure.

    16. The Solvent Accessible Reducing Ends Among the microcrystalline celluloses, they had the relatively same number of total of reducing ends, where approximately 66 % of those reducing ends are located at the surface. For the bacterial microcrystalline cellulose form (BMCC and AMCC), there are more reducing ends located at the surface of their structures. The 79% reducing ends exposed whereas the rest is still hidden in the crystalline structure.Among the microcrystalline celluloses, they had the relatively same number of total of reducing ends, where approximately 66 % of those reducing ends are located at the surface. For the bacterial microcrystalline cellulose form (BMCC and AMCC), there are more reducing ends located at the surface of their structures. The 79% reducing ends exposed whereas the rest is still hidden in the crystalline structure.

    17. Conclusion

    18. Question I would like to change the topic by asking the this question. Do the solvent accessible RE of cellulose behave/react the same as the RE of cellooligosaccharides in solution ? Since cellobiohydrolase activity is dependent on their cellulose chain ends interact with teh cellulose. Thus, it is important to know if these chain ends are analogous to these of oligosaccharide that are often used as a model substrate.I would like to change the topic by asking the this question. Do the solvent accessible RE of cellulose behave/react the same as the RE of cellooligosaccharides in solution ? Since cellobiohydrolase activity is dependent on their cellulose chain ends interact with teh cellulose. Thus, it is important to know if these chain ends are analogous to these of oligosaccharide that are often used as a model substrate.

    19. Experimental Approach I would like to change the topic by asking the this question. Do the solvent accessible RE of cellulose behave/react the same as the RE of cellooligosaccharides in solution ? Since cellobiohydrolase activity is dependent on their cellulose chain ends interact with teh cellulose. Thus, it is important to know if these chain ends are analogous to these of oligosaccharide that are often used as a model substrate.I would like to change the topic by asking the this question. Do the solvent accessible RE of cellulose behave/react the same as the RE of cellooligosaccharides in solution ? Since cellobiohydrolase activity is dependent on their cellulose chain ends interact with teh cellulose. Thus, it is important to know if these chain ends are analogous to these of oligosaccharide that are often used as a model substrate.

    20. Determine if the solvent accessible RE of cellulose behave/react the same as RE of cellooligosaccharides in solution ? What the RE look like ?

    21. Chemical system analyzed in here is described by the following reaction mechanism:

    22. The changes in concentrations of chemical species over time are computed by solving and initial value problem described by the following system of differential equations:

    23. Time-Course of Chemical Species for a Full Model

    24. Degradation Rate of Sodium Borohydride

    25. The changes in concentrations of chemical species over time are computed by solving and initial value problem described by the following system of differential equations:

    29. Reduction Rate of Solvent Accessible Reducing Ends

    30. Reduction Rate of Solvent Accessible Reducing Ends

    31. Results

    32. Conclusion

    34. Pectin Structure

    35. Pectin Structure

    36. Polygalacturonase and Pectate Lyase Activity

    38. Critical Parameters

    39. Reaction progress curves for the production of new reducing ends at different enzyme loads

    40. Relationship between enzyme load and amount of product generated at different extents of reaction

    41. Conclusion

    42. Acknowledgement

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