2.3 Computaional prediction for Sucrose isomerase thermostability
As starting point, the structure of WT and other mutants were modeled using the SI from Erwinia rhapontici NX5 (PDB: 4hph.1.A) as template with 74.10% sequence identity by SWISS-MODEL (https://www.swissmodel.expasy.org).[27]MolProbity[28] and PROCHECK[29] were applied for model evaluation, and evaluation results of WT are presented as Ramachandran plot (Fig S1). To identify the hot-spots for site-directed mutagenesis based on the ΔΔG change, FoldX 3.0 algorithm was utilized to estimate the folding free energy of SI. A standardized script written in python was performed to change all positions of the protein sequence to other 19 amino acids. The relative folding free energy changes (ΔΔG = ΔGMut − ΔGWT) was calculated after each residue was mutated into the other amino acids.