The Biocatalysis And Bioseparation Based On Chitosan With Near Neutral PH Responsive Property

pH-sensitive polymer materials are one kind of important "smart" materials, which have a Critical phase separation pH point(referred to CPP). In order to perform efficient bioaffinity capture and biocatalysis by using pH responsive polymer conjugant, polymers with near neutral CPP value and functional group for easy modification are highly desired. Chitosan, a natural polymer with pKa value of 6.3, has been recognized as a pH sensitive polymer since it has a large number of amino groups on its polymer backbone. In this paper, we used two methods to increase the CPP values of the chitosan polymer. One was to degrade the chitosan molecular to obtain the low molecular weight chitosan(LMWC), the other was to modify the chitosan with arginine, which has a relative higher pKa value. The method for preparation of LMWC and arginine midified chitosan, as well as the method for conjugation with protein and enzyme, was established. Finally, the conjugant of chitosan and modified chitosan with biological molecules were applied in the capature of trypsin inhibitors, protease activity determination and protein digestion for proteomic research.In chapter one, recent works related with pH-sensitive polymer materials, such as the method for changing the CPP value and the preparation of pH-sensitive polymer conjugant with biological molecules were reviewed.In chapter two, chitosan with different molecular weight was prepared by oxidative degradation method, in which hydrogen peroxide was employed as the oxidant. And the relationship between the molecular weight and CPP value was discussed. Results showed that, higher CCP value closing to neutral pH point could be obtained with the lower molecular weight chitosan.In chapter three, chitosan was modified with arginine by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) and N-hydroxysuccinimide(NHS) as coupling agents. The resulting product was characterized with FTIR and elementary analysis. The experimental data revealed that the CPP value of the modified CS is directly related with the subsitiude degree of arginine on chitosan backbond. The higher substitute degree lead to a increased CPP value of this conjugate.Chapter four presented several coupling methods between the chitosan and the biological molecules, including electrostatic assembly, covalent attachment and metal chelating coupling. Conjugation of the chitosan or modified chitosan with protein or enzyme was confirmed by Coomassie Brilliant Blue, enzyme activity assay, and Gel Permeation Chromatography(GPC).In chapter five, we discussed the effect of the Enzyme Inhibitors and BAPAN on the CS-Trypsin conjugate, and the sensitivity of Trypsin to the CS-ArgC-BSA conjugate. We found that Trypsin-CS conjugant could catalyze the degradation of protein in homogeneous phase when the pH was controlled in less than CPP value, and could be separated from solution by adjusting the pH to be higher than CPP. HPLC results show that the self degradation of trypsin was significantly inhibited and the interference from the self-degraded peptide could be eliminated in the peptide fragment mapping. Furthermore, the Arg-CS-BSA conjugant was sensitive to the Trypsin, and it provided a new method to detect the activity of Trypsin.