NDLI: Enhancing RGI lyase thermostability by targeted single point mutations

Content Provider	Springer Nature Link
Author	Silva, Inês R. Larsen, Dorte M. Jers, Carsten Derkx, Patrick Meyer, Anne S. Mikkelsen, Jørn D.
Copyright Year	2013
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.
Starting Page	9727
Ending Page	9735
Page Count	9
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	97
Issue Number	22
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2013-08-31
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Protein engineering Semi-rational Consensus approach Bacillus licheniformis Bacillus subtilis expression Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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