title: Improving thermal and detergent stability of Bacillus stearothermophilus neopullulanase by rational enzyme design.
abstract: Neopullulanase, a glycosyl hydrolase from Bacillus stearothermophilus (bsNpl), is a potentially valuable enzyme for starch and detergent industries. However, as the protein is not active at elevated temperatures and high surfactant concentrations, we aimed to increase its stability by rational enzyme design. Nine potentially destabilizing cavities were identified in the crystal structure of the enzyme. Based on computational predictions, these cavities were filled by residues with bulkier side chains. The five Asp46Glu, Val239Leu, Val404Leu, Ser407Thr and Ala566Leu exchanges resulted in a drastic stabilization of bsNpl against inactivation by heat and detergents. The catalytic activity of the variants was identical to the wild-type enzyme.
authors: Biochemistry Department, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors: Selin
authors: S
authors: Ece
authors: Biochemistry Department, Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany serap.evran@ege.edu.tr reinhard.sterner@biologie.uni-r.de.
authors: Serap
authors: S
authors: Evran
authors: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors: Jan-Oliver
authors: JO
authors: Janda
authors: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors: Rainer
authors: R
authors: Merkl
authors: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany serap.evran@ege.edu.tr reinhard.sterner@biologie.uni-r.de.
authors: Reinhard
authors: R
authors: Sterner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D006026:Glycoside Hydrolases; C058484:neopullulanase
keywords: enzyme design; neopullulanase; surfactant stability; thermal stability