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Enhancing subtilisin thermostability through a modified normalized B-factor analysis and loop-grafting strategy.

Heng Tang1, Ke Shi2, Cheng Shi3

  • 1Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China; School of Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, Jiangsu, China.

The Journal of Biological Chemistry
|October 17, 2019
PubMed
Summary

Researchers engineered a subtilisin E-S7 (SES7) peptidase for enhanced thermostability using a modified B-factor approach. This protein engineering strategy yielded a variant with a 7.3 °C higher melting temperature, showing industrial potential.

Keywords:
B-FIT strategybioinformaticscrystal structureloop graftingmolecular dynamics simulationspeptidaseprotein dynamicsprotein engineeringprotein motifprotein stabilityrational protein designsubtilisin E-S7thermostability

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Area of Science:

  • Protein Engineering
  • Biochemistry
  • Structural Biology

Background:

  • Improving protein thermostability is crucial for industrial applications.
  • Rational design often involves identifying and mutating unstable regions.
  • The B-factor method (B-FIT) is a common approach to identify flexible regions.

Purpose of the Study:

  • To enhance the thermostability of the subtilisin E-S7 (SES7) peptidase.
  • To develop a refined bioinformatics strategy for structure-based protein engineering.
  • To identify and introduce thermostable motifs into flexible regions of SES7.

Main Methods:

  • Utilized a normalized B-factor calculation to identify flexible regions, specifically loop 158-162.
  • Screened the Protein Data Bank for 29 thermostable motif sequences.
  • Employed iterative homologous modeling and site-directed mutagenesis to create chimera loops and SES7 variants.

Main Results:

  • Identified loop 158-162 as a critical flexible region in SES7.
  • Developed a thermostable SES7 variant (M5) with a 7.3 °C increase in melting temperature compared to wild-type (WT).
  • X-ray crystallography revealed M5 forms more hydrogen bonds than WT, consistent with molecular dynamics simulations.

Conclusions:

  • The modified B-FIT strategy is effective for rational protein engineering and enhancing thermostability.
  • The engineered SES7 variant M5 exhibits significantly improved thermal stability.
  • This approach holds promise for developing robust enzymes for industrial applications.