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Applied Microbiology and Biotechnology

, Volume 98, Issue 10, pp 4521–4531 | Cite as

Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

  • Inês R. Silva
  • Carsten Jers
  • Harm Otten
  • Christian Nyffenegger
  • Dorte M. Larsen
  • Patrick M. F. Derkx
  • Anne S. Meyer
  • Jørn D. Mikkelsen
  • Sine Larsen
Biotechnologically relevant enzymes and proteins

Abstract

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in T m of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects.

Keywords

Protein engineering Polysaccharide lyase family 11 RGI lyase Bacillus licheniformis Bacillus subtilis expression Crystal structure 

Notes

Acknowledgments

This study was supported by the Danish Strategic Research Council's Committee on Food and Health (FøSu) project “Biological Production of Dietary Fibers and Prebiotics” no. 2101-06-0067. We are grateful to Dorthe Boelskifte for help with the crystallization experiments. We thank the ESRF for synchrotron beamtime and for excellent support by the staff, DanScatt for travel support, and the University of Copenhagen Program of Excellence for funding.

Supplementary material

253_2013_5483_MOESM1_ESM.pdf (552 kb)
ESM 1 (PDF 552 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Inês R. Silva
    • 1
  • Carsten Jers
    • 1
  • Harm Otten
    • 2
  • Christian Nyffenegger
    • 1
  • Dorte M. Larsen
    • 1
  • Patrick M. F. Derkx
    • 3
  • Anne S. Meyer
    • 1
  • Jørn D. Mikkelsen
    • 1
  • Sine Larsen
    • 2
  1. 1.Center for Bioprocess Engineering, Department of Chemical and Biochemical EngineeringTechnical University of DenmarkKongens LyngbyDenmark
  2. 2.Department of ChemistryUniversity of CopenhagenCopenhagenDenmark
  3. 3.Chr. Hansen A/SHørsholmDenmark

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