title: Thermostability enhancement of an endo-1,4-β-galactanase from Talaromyces stipitatus by site-directed mutagenesis.
abstract: Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires development of thermostable enzymes. Talaromyces stipitatus was found to secrete endo-1,4-β-galactanase when grown on sugar beet pectin as sole carbon source. The mature protein contained 353 AA and the MW was estimated to 36.5 kDa. It was subjected to codon optimization and produced in Pichia pastoris in 2 l scale yielding 5.3 g. The optimal reaction condition for the endo-1,4-β-galactanase was determined to be 46 °C at pH 4.5 at which the specific activity was estimated to be 6.93 μmol/min/mg enzyme with half-lives of 13 and 2 min at 55 and 60 °C, respectively. For enhancement of the half-life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis of semi-rational design. Of these nine mutants, G305A showed half-lives of 114 min at 55 °C and 15 min at 60 °C, respectively. This is 8.6-fold higher than that of the TSGAL at 55 °C, whereas the other mutants displayed moderate positive to negative changes in their half-lives.
authors.affiliation: Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark.
authors.forename: Dorte M
authors.initials: DM
authors.lastname: Larsen
authors.forename: Christian
authors.initials: C
authors.lastname: Nyffenegger
authors.forename: Maria M
authors.initials: MM
authors.lastname: Swiniarska
authors.forename: Anders
authors.initials: A
authors.lastname: Thygesen
authors.forename: Mikael L
authors.initials: ML
authors.lastname: Strube
authors.forename: Anne S
authors.initials: AS
authors.lastname: Meyer
authors.forename: Jørn D
authors.initials: JD
authors.lastname: Mikkelsen
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D013379:Substrate Specificity; D032901:Talaromyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C089138:beta-galactanase