碩士 / 國立嘉義大學 / 生化科技學系研究所 / 101 / A unidentified Bacillus licheniformis trehalose-6-phosphate hydrolase ( BlTreA ) gene was cloned and heterologously expressed in Escherichia coli M15 cells. The over-expressed BlTreA was purified to apparent homogeneity by metal-affinity chromatography and its molecular mass was determined to be approximately 65.9 kDa. The temperature and pH optima for BlTreA were 30℃and 8.0, respectively. The enzyme hydrolyzed p-nitrophenyl-α-D-glucopyranoside ( pNPG ) and trehalose-6-phosphate efficiently, but it was inactive toward five other p-nitrophenyl derivatives. Steady-state kinetics with pNPG showed that BlTreA had a Km value of 5.4 mM and a kcat value of 31.9 s−1. Circular dichroism analysis revealed that the secondary structures of BlTreA did not altered by 5–10 % acetone and 10–20 % ethanol, whereas 5–10 % SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ∼0.14 M guanidine hydrochloride ( GdnHCl ) and reached the unfolded intermediates, [ GdnHCl ]0.5,N–I and [ GdnHCl ]0.5,I–U at 1.02 and 2.24 M, respectively. BlTreA was unfolded completely by 8 M urea with [ urea ]0.5,N–U of 4.98 M, corresponding to a free energy change of 4.29 kcal/mol for the N → U process. Moreover, the enzyme was unfolded by GdnHCl through a reversible pathway and the refolding reaction exhibited an intermediate state. According to the multiple sequences alignment and phylogenetic analysis, we can confirm that BlTreA is a member of glycoside hydrolase family 13.
Based on the sequence alignment of the selected Cl--dependent and independent glycoside hydrolase family 13 enzymes, amino acid residues Arg201, Asn327 and Tyr365 that might be responsible for the binding of BlTreA to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analysis. The mutant enzymes ( R201Q/E/K, N327Q/D/K, and Y365A/R ) and BlTreA were individually over-expressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 828.2 and 190.3 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With exception of Y365A, all mutant enzymes did not have the ability to bind chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes display a slight difference in the profiles of intrinsic tryptophan fluorescence. These results clearly indicate that Arg201 and Asn327 residues might play an essensital role in chloride binding of BlTreA.
| Identifer | oai:union.ndltd.org:TW/101NCYU5103021 |
| Creators | 莊紫婷 |
| Contributors | Ping-Lin Ong, Long-Liu Lin, 翁秉霖, 林榮流 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 125 |
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