Larsen, Dorte Møller5; Nyffenegger, Christian1; Swiniarska, Malgorzata Maria1; Thygesen, Anders1; Strube, Mikael Lenz3; Meyer, Anne S.1; Mikkelsen, Jørn Dalgaard1
1 Department of Chemical and Biochemical Engineering, Technical University of Denmark2 Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark3 National Veterinary Institute, Technical University of Denmark4 Section for Bacteriology, Pathology and Parasitology, National Veterinary Institute, Technical University of Denmark5 Department of Systems Biology, Technical University of Denmark
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 pro- duced 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 μ mol/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 mod- erate positive to negative changes in their half-lives.
Applied Microbiology and Biotechnology, 2015, Vol 99, Issue 10, p. 4245-4253
Protein engineering; Semi-rational design; Multiple alignment; GH53; Half-life