title: Characterization of a Novel α-l-Arabinofuranosidase from Ruminococcus albus 7 and Rational Design for Its Thermostability.
abstract: An α-l-arabinofuranosidase (Abf) encoding gene was obtained via genomic mining from a Ruminococcus albus strain. The specific activity of this GH 51 Abf was 73.3 U/mg at pH 6.0 and 50 °C. The modification of Abf, aimed at improving thermostability, was performed through different strategies. Structure-based rational design using the PoPMuSiC and the Enzyme Thermal Stability System (ETSS) predicted thermal stability of Abf and enhanced the half-life of thermal inactivation (t1/2) at 50 °C for K208W more than 11.1 times versus the wild-type (WT). Sequence-based rational design was also conducted by substituting histidine with lysine at various sites. Among eight mutants, the t1/2at 50 °C of H337K was prolonged by 5.0-fold, and the specific activity of this mutant was increased to 121.8 U/mg. In addition, the mutant H337K was utilized with some enzymes to extract pectin from apple pomace. The enzymatically produced pectin got less moisture and ash, milder pH, and higher viscosity than its acid-extracted counterpart, indicating that Abf has an application prospect in pectin production.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology , Shanghai 200237, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology , Shanghai 200237, People's Republic of China.
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology , Shanghai 200237, People's Republic of China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology , Shanghai 200237, People's Republic of China.
authors.forename: Lujia
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Food Science and Technology, Tokyo University of Marine Science and Technology , Tokyo 108-8477, Japan.
authors.forename: Lei
authors.initials: L
authors.lastname: Du
authors.affiliation: Department of Food Science and Technology, Tokyo University of Marine Science and Technology , Tokyo 108-8477, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Matsukawa
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology , Shanghai 200237, People's Republic of China.|Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB) , Shanghai 200237, People's Republic of China.
authors.forename: Jingli
authors.initials: J
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology, School of Biotechnology, East China University of Science and Technology , Shanghai 200237, People's Republic of China.|Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB) , Shanghai 200237, People's Republic of China.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010368:Pectins / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D045857:Ruminococcus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D010368:Pectins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: pectin; rational design; site-directed mutagenesis; thermostability; α-l-arabinofuranosidase