TY - JOUR
T1 - Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations
AU - da Silva, Ines Isabel Cardoso Rodrigues
AU - Jers, Carsten
AU - Otten, Harm
AU - Nyffenegger, Christian
AU - Larsen, Dorte Møller
AU - Derkx, Patrick M.F.
AU - Meyer, Anne S.
AU - Mikkelsen, Jørn Dalgaard
AU - Larsen, Sine
PY - 2014
Y1 - 2014
N2 - 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 Tm 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.
AB - 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 Tm 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.
KW - Bacillus licheniformis
KW - Bacillus subtilis expression
KW - Crystal structure
KW - Polysaccharide lyase family 11
KW - Protein engineering
KW - RGI lyase
U2 - 10.1007/s00253-013-5483-8
DO - 10.1007/s00253-013-5483-8
M3 - Journal article
C2 - 24419797
AN - SCOPUS:84900808594
SN - 0175-7598
VL - 98
SP - 4521
EP - 4531
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 10
ER -