The Structural Analysis And Molecular Modification Of Starch Debranching Enzyme CDS1-3

Amylopectin degradation is widely studied by researchers.Starch debranching enzyme is a group of enzymes that hydrolyze alpha-1,6-glucosidic bond. In this work, the mechanism of amylopectin hydrolyzed by starch debranching enzyme was studied and this will provided a new strategy for the research of starch debranching enzyme.A gene cdsl-3 (1764 bp) was obtained from a DNA library and recombinant Escherichia coli DH5a/pSE3% 0-cdsl-3 was constructed. The starch debranching enzyme encoded by gene cds1-3, termed CDS 1-3, was purified by Ni affinity chromatography and ion exchange chromatography. Enzyme function characterization was performed for CDS 1-3. Results revealed that optimal temperature and pH of CDS1-3 were 50℃ and 5.5 respectively. The preferred substrates of recombinant enzyme CDS 1-3, in the descending order, were alpha-cyclodextrin, pullulan and tapioca starch. Vmax and Km to alpha-cyclodextrin were 19.45 μmol/(min*mg) and 1.63 mg/L respectively. and Km to soluble starch were 20.23 μmol/(min*mg) and 39.52 mg/L respectively. Vmax and Km to amylopectin were 37.95 μmol/(min*mg) and 184.9 mg/L respectively. CDS 1-3 and bacterial α-amylase produced by Aladdin had negative synergistic effect to subtrates with low molacular weight and positive synergistic effect to amylopectin.CDS 1-3 shows 94.7% homology to starch debranching enzyme produced by Thermus sp. IM6501. However, degradation activity of these two enzymes shows large differences. By comparing of the amino acid sequence and protein structure, three position (Glu66, Pro48, Phe289) were choosed for mutation.Three mutants (E66G, P48H, F289A) were obtained by PCR. Out of these three mutants, E66G showed catalytic activity. Compared with wild type enzyme, mutational enzyme showed higher activity on degradation of the substrates with high molecular weight. These results showed that E66 is one of key amino acids on degradation of macromolecule substrates.