1 Department of Chemical and Biochemical Engineering, Technical University of Denmark2 Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark3 Department of Systems Biology, Technical University of Denmark4 Department of Biotechnology, Technical University of Denmark5 Institute for Product Development, Technical University of Denmark6 Technical University of Denmark7 Infection Microbiology, Department of Biotechnology and Biomedicine, Technical University of Denmark8 Chr. Hansen A/S
Rhamnogalacturonan I lyase (RGI lyase) (EC 4.2.2.-) catalyzes the cleavage of rhamnogalacturonan I in pectins by β-elimination. In this study the thermal stability of a RGI lyase (PL 11) originating from Bacillus licheniformis DSM 13/ATCC14580 was increased by a targeted protein engineering approach involving single amino acid substitution. Nine individual amino acids were selected as targets for site-saturated mutagenesis by the use of a predictive consensus approach in combination with prediction of protein mutant stability changes and B-factor iteration testing. After extensive experimental verification of the thermal stability of the designed mutants versus the original wild-type RGI lyase, several promising single point mutations were obtained, particularly in position Glu434 on the surface of the enzyme protein. The best mutant, Glu434Leu, produced a half-life of 31 min at 60 °C, corresponding to a 1.6-fold improvement of the thermal stability compared to the original RGI lyase. Gly55Val was the second best mutation with a thermostability half-life increase of 27 min at 60 °C, and the best mutations following were Glu434Trp, Glu434Phe, and Glu434Tyr, respectively. The data verify the applicability of a combinatorial predictive approach for designing a small site saturation library for improving enzyme thermostability. In addition, new thermostable RGI lyases suitable for enzymatic upgrading of pectinaceous plant biomass materials at elevated temperatures were produced.
Applied Microbiology and Biotechnology, 2013, Vol 97, Issue 22, p. 9727-9735
Protein engineering; Semi-rational; Consensus approach; Bacillus licheniformis; Bacillus subtilis expression