title: Enhancing RGI lyase thermostability by targeted single point mutations.
abstract: 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.
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Inês R
authors.initials: IR
authors.lastname: Silva
authors.forename: Dorte M
authors.initials: DM
authors.lastname: Larsen
authors.forename: Carsten
authors.initials: C
authors.lastname: Jers
authors.forename: Patrick
authors.initials: P
authors.lastname: Derkx
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010368:Pectins / Q000378:metabolism*; D017354:Point Mutation; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055550:Protein Stability; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; C042491:rhamnogalacturonan I; D010368:Pectins; D011133:Polysaccharide-Lyases