Research On Hydrophobic Force Of Deacetylated Konjac Glucomannan In The Process Of Self-crimping And Aggregating And Its Application On Dna Gel Electrophoresis

The evaluation based on the role and contribution of other sub-interaction except for hydrogen bonding interaction in neutral polysaccharide Sol-gel system is the basic scientific question in the carbohydrate chemistry field. The driving and maintaining force of deaccetylated konjac glucomannan in self crimping, aggregating and sol-gel transition is still a matter of controversy in konjac science. To find the high value added and reasonable utilization way of konjac whose price is increasing year afer year is the inevitable requirements of industrial upgrading.In the present study, viscometry and TPA analysis was employed to identify the presence of hydrophobic interactions in the process of self crimping, aggregating and gelling of d-KGM from extremely dilute solution to high concentrate solution. The fluorescence spectra, circular dichroism spectra(CD) and ultraviolte visible spectra(UV-Vis) of congo red which is a kind of fluorescent probes was investigated to gain further insight into it. Pyrene fluorescence probe technology combined with atomic force microscopy (AFM) was primary used to confirm and roughly determinate the presence of hydrophobic interactions. In addition, deacetylated konjac glucomannan as the composition of gel electrophoresis matrix in bioseparation was attempted in order to explore the effect of hydrophobic interactions on bioseparation and also lay thefoundation for developing a novel kind of low cost, low electroendosmosis and highefficient gel electrophoresis matrix. The main contents and conclusions in this work arelisted as follows:1. With increaseing concentration of NaCl, CaCl₂, Na₂SO₄, NaNO₃auqeous solution, the size of the d-KGM molecule was reduced, and the reducing rate was Cl^->SO₄^2->NO₃^-, Ca²⁺>Na⁺. The intrinsic viscosity of d-KGM was increased slightly in the case of urea with low concentration due to the fact that hydrogen bonding was destroyed by urea, whereas was decreased rapidly with high concentration, which demonstrated that self crimping of d-KGM molecular chain was not only resulted in hydrogen bonding interaction. The obvious increase of apparent size of d-KGM in extremely SDS aqueous solution suggested that hydrophobic micro-area existed in the self crimp chain of d-KGM and co-aggregated with hydrophobic group of SDS.2. The results that the hardness of gel decreased strongly because of hydrogen bonding broken by urea, yet the springiness and cohesiveness was changed slightly indicated maintaining force of gel network structure (hard structure) was another stronger force which is different from hydrogen bonds. The rising of hardness, springness, cohesiveness and resilience of d-KGM gel added SDS with lower than the 0.08 mol/L demonstrated hydrophobic micro-area involving in SDS in the hard structure strengthened the hydrophobic interactions. The hardness and cohesiveness of gel were increased significantly when the temperature increased and achieved the highest point at 65℃. This performance is similar to the theory of protein hydrophobic interactions and implied that hydrogen bonds is not the main force to maintain gel network.3. Dynamic contact concentration (Cs) of KGM and d-KGM is 0.55g/L and 0.65g/L, moreover the entangling concentration(Ce) is 5.60g/Land 4.50g/L, respectively. The CD and fluorescence spectral analysis confirmed the increasing of unsymmetry and notable conformational transition from disordered to some little ordered structure of KGM after deacelyzation. From the UV-VIS analysis, the ordered structure of d-KGM was remarkably different from helical structure. It is expressed that hydrophobic interactions meight play an important role again.4. It was confirmed that the presence of hydrophobic interactions, since I₁/I₃ decreased as the d-KGM concentration increased in the extremely dilute and semidilute solution adding pyhene probe. Howerer, I₁/I₃ decreased only a little due to the absence of hydrophobic groups on the molecular main chain of d-KGM. Na₂SO₄ and NaNO₃ (or NaCl) could be considered as the deaggregator and enhancing aggregator of d-KGM respectively. As the concentration of urea aqueous solution was beyond upon 1.00 mol/L, hydrophobic interactions between d-KGM molecules was weakened. Furthmore, co-aggregateing between SDS and d-KGM molecule accelerated the forming of hydrophobic micro-area.5. From the image of atomic force microscope, KGM occurred intensely self-crimp afer total deacetylation in form of stable conical structure; Pyrene probe was on the top of conical structure and hydrophobic interactions promoted the solubility of pyhene probe; The phenomenon that d-KGM molecules haphazard packed, but fair parts still maintained the spherical configuration indicated that hydrophobic interactions was not destroyed by low concentrate urea aqueous solution. The performance that SDS and d-KGM could form complex which arrayed orderly and maintained sphere, and the dimension of aggregation was larger and the height was 4-5nm reflected hydrophobic aggregation between SDS and d-KGM molecule.6. The electroendosmosis of AG gel added the d-KGM was reduced obviously and is almost zero, while the additive amount of the d-KGM achieved 0.75%. The uniformity of mixture gel after electrophoresis was greatly improved than AG gel. Increasing rate of diffusion coefficient in mixture gel declined and differed from the rules in AG gel. The slope of DNA marker mobility rised greatly and clear high resolution electrophoresis image was obtained in short time after adding d-KGM. Howerer, the relationship between the chang of resolution and addition of d-KGM was not remarkabely. It was implied that the mechanism of DNA separation in d-KGM gel is unlike in AG gel for the special properties of hydrophobic interactions.