| I. A new general method to immobilize proteins/enzymes, with easy operation and little activity loss, was developed. The protein immobilized by this method can be used in aqueous solutions without significant leaching, or it can be dehydrated and used as a biocatalyst in organic media. In this work, I demonstrated the applicability of this method for alpha-chymotrypsin, subtilisin and hemoglobin. First, the enzymatic activity of alpha-chymotrypsin and subtilisin both in immobilized and free form was studied. The transesterification activity of the two embedded enzymes in organic solvent compared to those of the free enzymes suspended in organic solvent, increased by a factor of more than 300. The hydrolytic activity of the enzyme preparations in aqueous solution was up to 76% of that of the free enzyme in aqueous solution. Further, the thermostability of those protein preparations was also investigated. The half-lives of the two immobilized enzymes at 100°C in octane were almost quadrupled compared to free enzymes, and even more dramatically improved compared to free enzymes in aqueous solution.;II. Arginine repressor (ArgR) of E. coli, the product of the negatively autoregulated argR gene, controls all 12 genes of the arginine regulon. Here, the affinity, specificity, cooperativity, stoichiometry and L-arginine sensitivity for binding of ArgR and ArgRN(1--78) to DNA constructs with different ARG box arrangements, including half-box, one-box, and two-box with 3 by spacing were studied. The binding stoichiometry of ArgR (hexamer) with oligonucleotides containing two, one or half ARG box was determined to be 1:1, 1:3 or 1:6 respectively. The binding experiment results suggest that half ARG box is the minimum unit for ArgR/DNA binding, i.e. the minimal unit ArgR uses to involve in protein/DNA interaction is monomer.;The bending studies of the ARG box-containing DNA fragments showed that the DNA fragment containing one ARG box was bent 35° upon ArgR binding, compared to 85° bent for DNA fragment containing two ARG boxes upon ArgR binding. Also, the DNA fragment containing two ARG box can be bent 42° upon ArgRN binding. |
