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

Publication ,  Journal Article
Acharya, P; Rajakumara, E; Sankaranarayanan, R; Rao, NM
Published in: J Mol Biol
August 27, 2004

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.

Duke Scholars

Published In

J Mol Biol

DOI

ISSN

0022-2836

Publication Date

August 27, 2004

Volume

341

Issue

5

Start / End Page

1271 / 1281

Location

Netherlands

Related Subject Headings

  • Protein Structure, Tertiary
  • Protein Denaturation
  • Point Mutation
  • Models, Molecular
  • Lipase
  • Hot Temperature
  • Evolution, Molecular
  • Enzyme Stability
  • Crystallography, X-Ray
  • Biochemistry & Molecular Biology
 

Citation

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Acharya, P., Rajakumara, E., Sankaranarayanan, R., & Rao, N. M. (2004). Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase. J Mol Biol, 341(5), 1271–1281. https://doi.org/10.1016/j.jmb.2004.06.059
Acharya, Priyamvada, Eerappa Rajakumara, Rajan Sankaranarayanan, and Nalam M. Rao. “Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.J Mol Biol 341, no. 5 (August 27, 2004): 1271–81. https://doi.org/10.1016/j.jmb.2004.06.059.
Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM. Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase. J Mol Biol. 2004 Aug 27;341(5):1271–81.
Acharya, Priyamvada, et al. “Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase.J Mol Biol, vol. 341, no. 5, Aug. 2004, pp. 1271–81. Pubmed, doi:10.1016/j.jmb.2004.06.059.
Acharya P, Rajakumara E, Sankaranarayanan R, Rao NM. Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase. J Mol Biol. 2004 Aug 27;341(5):1271–1281.
Journal cover image

Published In

J Mol Biol

DOI

ISSN

0022-2836

Publication Date

August 27, 2004

Volume

341

Issue

5

Start / End Page

1271 / 1281

Location

Netherlands

Related Subject Headings

  • Protein Structure, Tertiary
  • Protein Denaturation
  • Point Mutation
  • Models, Molecular
  • Lipase
  • Hot Temperature
  • Evolution, Molecular
  • Enzyme Stability
  • Crystallography, X-Ray
  • Biochemistry & Molecular Biology