PMC

Influence of single, double, and triple disulfide bridges on the optimum temperature.

Influence of pH on the activity (A) and stability (B) of wild-type and mutant enzymes.

Circular dichroism (CD) (A and B) and fluorescence spectroscopy (C and D) of wild-type and mutant enzymes.

The half-life (t_1/2) of wild-type AmyK and its mutants at 60°C. The purified enzymes were incubated at 60°C for designed periods, followed by the standard activity assay.

Model structure of mutant P35C-G426C/G116C-Q120C/R426C-M480C. The model structure of this mutant was constructed with the crystal structure of AmyB (3bc9) as a template. The α helices and β sheets are shown in red and cyan, respectively. The catalytic residues Asp 248, Glu 278, and Asp 340 are shown in green according to the CPK (Corey-Pauling-Koltun) representation scheme. The yellow sticks indicate the mutation sites.

Model structure of AmyK and local model of mutation sites and catalytic residues in AmyK. The model structure of AmyK was constructed with the crystal structure of AmyB (3bc9) as a template. The α helices and β sheets are shown in red and cyan, respectively. The catalytic residues Asp 248, Glu 278, and Asp 340 are shown in green according to the CPK (Corey-Pauling-Koltun) representation scheme. The yellow ball-and-stick structures indicate the mutation sites.

Change of the salt bridge around active sites of mutant G116C-120C. The α helices and β sheets are shown in red and cyan, respectively. The catalytic residues Asp 248, Glu 278, and Asp 340 are shown in yellow ball-and-stick representation. The blue sticks are the mutation sites. The pink sticks are residues forming a salt bridge around the active sites, and the dark dotted line is the salt bridge. The green sticks are the residues Asp 195 and Lys 202 before mutation. (A) Salt bridge around active sites of wild-type AmyK. (B) Change of salt bridge around active sites of the mutant.

Local hydrogen bonding network of mutant P35C-G426C. The hydrogen bonds were displayed with Discovery Studio 2.5. The α helices and β sheets are shown in red and cyan, respectively. The catalytic residues Asp 248, Glu 278, and Asp 340 are shown in yellow ball-and-stick representations. The blue sticks are the mutation sites. The pink sticks are the changed residues that form hydrogen bonding before and after mutation. The dark dotted line is the hydrogen bond. (A) Wild type; (B) P35C-G426C.