BACKGROUND AND ABJECTIVE: Site directed mutagenesis in protein flexible regions is an efficient protein engineering method to increase thermal stability. In order to have wider industrial applications of enzymes such as Cel5E of Clostridium thermocellum, high thermal stability is necessary. MATERIALS AND METHODS: Modeling of native CEL5E and mutant enzyme was done based on homology modeling using swissmodel server. The evaluation of the predicted models was done based on GMQE and QMEAN4 parameters and also in the SAVES server. Flexible regions in CEL5E were predicted using molecular dynamic simulation methods and GROMACS. The binding affinity of native and mutant enzyme to cellulose substrate was evaluated by AUTODOCK VINA. After introducing the flexible regions, the mutagenesis candidates were predicted by SDM, CUPSAT, POP MUSIC, HOT MUSIC and FIREPROT servers. RESULTS AND DISCUSSION: The results showed that in CEL5E enzyme, the residues in position 58-66 have the most flexibility and the highest RMSF (root mean square fluctuation) value. In this region, R63H mutation was predicted and introduced as the best enzyme stabilizing mutation based on ΔΔG and ΔTm. CONCLUSION: The results of this research led to the introduction of hotspots, th introduction of mutations producing commercial enzyme strains and enzyme isoforms with the best stability. By performing advanced computational tests in quasi-real conditions, it was possible to obtain the best novel isoforms that are more capable than the existing native isoforms.

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