Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.


Journal Article

Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.

Full Text

Duke Authors

Cited Authors

  • Acharya, P; Rajakumara, E; Sankaranarayanan, R; Rao, NM

Published Date

  • August 27, 2004

Published In

Volume / Issue

  • 341 / 5

Start / End Page

  • 1271 - 1281

PubMed ID

  • 15321721

Pubmed Central ID

  • 15321721

International Standard Serial Number (ISSN)

  • 0022-2836

Digital Object Identifier (DOI)

  • 10.1016/j.jmb.2004.06.059


  • eng

Conference Location

  • England