title: Thermostabilization of Pichia stipitis xylitol dehydrogenase by mutation of structural zinc-binding loop.
abstract: Xylitol dehydrogenase from Pichia stipitis (PsXDH) is one of the key enzymes for the bio-ethanol fermentation system from xylose. Previously, we constructed the C4 mutant (S96C/S99C/Y102C) with enhanced thermostability by introduction of structural zinc. In this study, for further improvement of PsXDH thermostability, we constructed the appropriate structural zinc-binding loop by comparison with other polyol dehydrogenase family members. A high thermostability of PsXDH was obtained by subsequent site-directed mutagenesis of the structural zinc-binding loop. The best mutant in this study (C4/F98R/E101F) showed a 10.8 degrees C higher thermal transition temperature (T(CD)) and 20.8 degrees C higher half denaturation temperature (T(1/2)) compared with wild-type.
authors.affiliation: Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Japan.
authors.forename: Narayana
authors.initials: N
authors.lastname: Annaluru
authors.forename: Seiya
authors.initials: S
authors.lastname: Watanabe
authors.forename: Seung Pil
authors.initials: SP
authors.lastname: Pack
authors.forename: Ahmed Abu
authors.initials: AA
authors.lastname: Saleh
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Kodaki
authors.forename: Keisuke
authors.initials: K
authors.lastname: Makino
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D050542:D-Xylulose Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology*; D011487:Protein Conformation; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D050542:D-Xylulose Reductase; D015032:Zinc