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Enhancing RGI lyase thermostability by targeted single point mutations.
- Source :
-
Applied Microbiology & Biotechnology . Nov2013, Vol. 97 Issue 22, p9727-9735. 9p. - Publication 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01757598
- Volume :
- 97
- Issue :
- 22
- Database :
- Academic Search Index
- Journal :
- Applied Microbiology & Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 91734068
- Full Text :
- https://doi.org/10.1007/s00253-013-5184-3