Novel Barley (1→3,1→4)-β-Glucan Endohydrolase Alleles Confer Increased Enzyme Thermostability

详细信息    查看全文

• 作者：Juanita C. LauerKuok Yap ; Suong Cu ; Rachel A. Burton ; Jason K. Eglinton
• 刊名：Journal of Agricultural and Food Chemistry
• 出版年：2017
• 出版时间：January 18, 2017
• 年：2017
• 卷：65
• 期：2
• 页码：421-428
• 全文大小：391K
• ISSN：1520-5118

文摘

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 T₅₀ value, 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.