title: Novel Barley (1→3,1→4)-β-Glucan Endohydrolase Alleles Confer Increased Enzyme Thermostability.
abstract: Barley (1→3,1→4)-β-glucan endohydrolases (β-glucanases; EI and EII) are primarily responsible for hydrolyzing high molecular weight (1→3,1→4)-β-glucans (β-glucan) during germination. Incomplete endosperm modification during malting results in residual β-glucan that can contribute to increased wort viscosity and beer chill haze. Four newly identified forms of EI and EII and the reference enzymes EI-a and EII-a were expressed in Escherichia coli, and the recombinant proteins were characterized for enzyme kinetics and thermostability. EI and EII variants that exhibited higher residual β-glucanase activity than EI-a and EII-a after heat treatment also exhibited increased substrate affinity and decreased turnover rates. The novel EII-l form exhibited significantly increased thermostability compared with the reference EII-a when activity was measured at elevated temperature. EII-l exhibited a T50value, which indicates the temperature at which 50% of β-glucanase activity remains, 1.3 °C higher than that of EII-a. The irreversible thermal inactivation difference between EII-a and EII-l after 5 min of heat treatment at 56 °C was 11.9%. The functional significance of the three amino acid differences between EII-a and EII-l was examined by making combinatorial mutations in EII-a using site-directed mutagenesis. The S20G and D284E amino acid substitutions were shown to be responsible for the increase in EII-1 thermostability.
authors.affiliation: School of Agriculture, Food & Wine, The University of Adelaide , Waite Campus, PMB 1, Glen Osmond, South Australia 5064, Australia.
authors.forename: Juanita C
authors.identifier: http://orcid.org/0000-0002-5857-2461
authors.initials: JC
authors.lastname: Lauer
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of Agriculture, Food & Wine, The University of Adelaide , Waite Campus, PMB 1, Glen Osmond, South Australia 5064, Australia.
authors.forename: Kuok
authors.initials: K
authors.lastname: Yap
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of Agriculture, Food & Wine, The University of Adelaide , Waite Campus, PMB 1, Glen Osmond, South Australia 5064, Australia.
authors.forename: Suong
authors.initials: S
authors.lastname: Cu
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of Agriculture, Food & Wine, The University of Adelaide , Waite Campus, PMB 1, Glen Osmond, South Australia 5064, Australia.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Burton
authors.affiliation: School of Agriculture, Food & Wine, The University of Adelaide , Waite Campus, PMB 1, Glen Osmond, South Australia 5064, Australia.
authors.forename: Jason K
authors.initials: JK
authors.lastname: Eglinton
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C028020:licheninase
keywords: catalytic efficiency; thermostability; wild barley; β-glucanase