Effect of mutations on the thermostability of Aspergillus aculeatus β-1,4-galactanase.
New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions were experimentally validated by measurements of the melting temperature (Tm ) by differential scanning calorimetry. We found that the Tm is elevated by 1.1 °C for G306A, slightly increased (in the range of 0.34 to 0.65 °C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability. Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also investigated for the wild-type and three variants and found to be biphasic. The half-lives of thermal inactivation were approximately doubled in G306A, unchanged for D182N and, disappointingly, a lot lower for D58N. In conclusion, this study tests a new method for estimating Tm changes for mutants, adds to the available data on the effect of substitutions on protein thermostability and identifies an interesting thermostabilizing mutation, which may be beneficial also in other galactanases.
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Søs
S
Torpenholt
Novozymes A/S, Smørmosevej 25, 2880 Bagsværd, Denmark.
Leonardo
L
De Maria
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Mats H M
MH
Olsson
Novozymes A/S, Smørmosevej 25, 2880 Bagsværd, Denmark.
Lars H
LH
Christensen
Novozymes A/S, Smørmosevej 25, 2880 Bagsværd, Denmark.
Michael
M
Skjøt
NSM, Research Unit for Functional Biomaterials, University of Roskilde, Universitetsvej 1, 4000 Roskilde, Denmark.
Peter
P
Westh
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Jan H
JH
Jensen
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark.
Leila
L
Lo Leggio
AZCL-galactan, azurine-crosslinked galactan; AaGal, β-1,4-galactanase from Aspergillus aculeatus; CAZY, carbohydrate active enzyme database; Computational prediction; DSC, differential scanning calorimetry; GH53; MtGal, β-1,4-galactanase from Myceliophthora thermophila; Protein design; Thermostability; Tm, melting temperature; TsGal, Talaromyces stipitatus galactanase; WT, wild type; β-1,4-galactanase