Improving the thermal stability of Cel۵E of Clostridium thermocellum by protein engineering of flexible regions
Publish place: 24th International Congress of Microbiology of Iran
Publish Year: 1402
نوع سند: مقاله کنفرانسی
زبان: English
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شناسه ملی سند علمی:
MEDISM24_053
تاریخ نمایه سازی: 6 اسفند 1402
Abstract:
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 Cel۵E of Clostridium thermocellum, high thermal stability is necessary.MATERIALS AND METHODS: Modeling of native CEL۵E and mutant enzyme was done based on homology modeling using swissmodel server. The evaluation of the predicted models was done based on GMQE and QMEAN۴ parameters and also in the SAVES server. Flexible regions in CEL۵E 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 CEL۵E enzyme, the residues in position ۵۸-۶۶ have the most flexibility and the highest RMSF (root mean square fluctuation) value. In this region, R۶۳H 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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Authors
Nazanin Gholampour-Faroji
Biotechnology Department, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
Jafar Hemmat
Biotechnology Department, Iranian Research Organization for Science and Technology (IROST), Tehran, Iran
Aliakbar Haddad-Mashadrizeh
Industrial Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
Ahmad Asoodeh
Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran