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Type: Journal article
Title: Novel barley (1→3,1→4)-β-glucan endohydrolase alleles confer increased enzyme thermostability
Other Titles: Novel barley (1->3,1->4)-beta-glucan endohydrolase alleles confer increased enzyme thermostability.
Author: Lauer, J.
Yap, K.
Cu, S.
Burton, R.
Eglinton, J.
Citation: Journal of Agricultural and Food Chemistry, 2017; 65(2):421-428
Publisher: American Chemical Society
Issue Date: 2017
ISSN: 0021-8561
Statement of
Juanita C. Lauer, Kuok Yap, Suong Cu, Rachel A. Burton, Jason K. Eglinton
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 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.
Keywords: β-glucanase; thermostability; catalytic efficiency; wild barley
Rights: © 2016 American Chemical Society
DOI: 10.1021/acs.jafc.6b04287
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Appears in Collections:Agriculture, Food and Wine publications
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