Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations
- 553 Downloads
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.
KeywordsProtein engineering Polysaccharide lyase family 11 RGI lyase Bacillus licheniformis Bacillus subtilis expression Crystal structure
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.
- Holck J, Lorentzen A, Vigsnæs LK, Licht TR, Mikkelsen JD, Meyer AS (2011) Feruloylated and nonferuloylated arabino-oligosaccharides from sugar beet pectin selectively stimulate the growth of Bifidobacterium spp. in human fecal in vitro fermentations. J Agric Food Chem 59:6511–6519PubMedCrossRefGoogle Scholar
- Jensen JL, Mølgaard A, Navarro Poulsen JC, Harboe MK, Simonsen JB, Lorentzen AM, Hjernø K, van den Brink JM, Qvist KB, Larsen S (2013) Camel and bovine chymosin: the relationship between their structures and cheese-making properties. Acta Crystallogr D Biol Crystallogr 69:901–913CrossRefGoogle Scholar
- Michalak M, Thomassen LV, Roytio H, Ouwehand AC, Meyer AS, Mikkelsen JD (2012) Expression and characterization of an endo-1,4-β-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans. Enzyme Microb Technol 50:121–129PubMedCrossRefGoogle Scholar
- Xiao Z, Bergeron H, Grosse S, Beauchemin M, Garron M, Shaya D, Sulea T, Cygler M, Lau PCK (2008) Improvement of the thermostability and activity of a pectate lyase by single amino acid substitutions, using a strategy based on melting-temperature-guided sequence alignment. Appl Environ Microbiol 74:1183–1189PubMedCentralPubMedCrossRefGoogle Scholar