caa a22 4500 605502315 CHVBK 20210128100605.0 cr unu---uuuuu 210128e20150501xx s 000 0 eng 10.1007/s00253-014-6244-z doi (NATIONALLICENCE)springer-10.1007/s00253-014-6244-z Thermostability enhancement of an endo-1,4-β-galactanase from Talaromyces stipitatus by site-directed mutagenesis [Elektronische Daten] [Dorte Larsen, Christian Nyffenegger, Maria Swiniarska, Anders Thygesen, Mikael Strube, Anne Meyer, Jørn Mikkelsen] 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 36.5kDa. It was subjected to codon optimization and produced in Pichia pastoris in 2l scale yielding 5.3g. The optimal reaction condition for the endo-1,4-β-galactanase was determined to be 46°C at pH4.5 at which the specific activity was estimated to be 6.93μmol/min/mg enzyme with half-lives of 13 and 2min 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 114min at 55°C and 15min 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. Springer-Verlag Berlin Heidelberg, 2014 Protein engineering nationallicence Semi-rational design nationallicence Multiple alignment nationallicence GH53 nationallicence Half-life nationallicence Larsen Dorte Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut Nyffenegger Christian Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut Swiniarska Maria Dako Denmark A/S, Produktionsvej 42, 2600, Glostrup, Denmark aut Thygesen Anders Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut Strube Mikael Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut Meyer Anne Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut Mikkelsen Jørn Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4245-4253 0175-7598 99:10<4245 2015 99 253 https://doi.org/10.1007/s00253-014-6244-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6244-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Larsen Dorte Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut NATIONALLICENCE 700 1- Nyffenegger Christian Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut NATIONALLICENCE 700 1- Swiniarska Maria Dako Denmark A/S, Produktionsvej 42, 2600, Glostrup, Denmark aut NATIONALLICENCE 700 1- Thygesen Anders Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut NATIONALLICENCE 700 1- Strube Mikael Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut NATIONALLICENCE 700 1- Meyer Anne Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut NATIONALLICENCE 700 1- Mikkelsen Jørn Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 229, 2800, Kongens Lyngby, Denmark aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4245-4253 0175-7598 99:10<4245 2015 99 253