机构地区:[1]Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Shiga, Japan [2]Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan [3]Department of Bioscience and Biotechnology, Fukui Prefectural University, Fukui, Japan
出 处:《Advances in Biological Chemistry》2020年第6期157-171,共15页生物化学进展(英文)
摘 要:<span style="font-family:Verdana;">L-Theanine (</span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamylethylamide) is a naturally occurring amino acid derivative known to have several beneficial physiological effects as a diet supplement, and to give an umami taste when used as a food additive. The compound is industrially produced by </span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamyltranspeptidase from </span><i><span style="font-family:Verdana;">Pseudomonas nitroreducens</span></i><span style="font-family:Verdana;"> (PnGGT). Using recombinant PnGGT, we have shown previously that Trp385, Phe417, and Trp525 are key amino acid residues for recognition of acceptor substrates at the PnGGT active site. Here, we demonstrate that a recombinant W525D mutant of PnGGT produces L-theanine from ethylamine and L-glutamine more efficiently than wild-type PnGGT, attributable to an increased ratio of transfer activity to hydrolysis activity. An efficient production of L-theanine was achieved by immobilizing </span><i><span style="font-family:Verdana;">Escherichia coli</span></i><span style="font-family:Verdana;"> cells expressing the W525D PnGGT mutant (</span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D) using 2% alginate as the supporting material. The highest L-theanine production using immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D, representing a conversion rate of 90%, w</span><span style="font-family:Verdana;">as</span><span style="font-family:Verdana;"> achieved in optimal reaction conditions of pH 10, 40<span style="white-space:nowrap;">°</span></span><span style="font-family:;" "=""><span><span style="font-family:Verdana;">C, and a substrate molar ratio of L-glutamine to ethylamine of 1:10. The immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana<span style="font-family:Verdana;">L-Theanine (</span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamylethylamide) is a naturally occurring amino acid derivative known to have several beneficial physiological effects as a diet supplement, and to give an umami taste when used as a food additive. The compound is industrially produced by </span><i><span style="font-family:Verdana;">γ</span></i><span style="font-family:Verdana;">-glutamyltranspeptidase from </span><i><span style="font-family:Verdana;">Pseudomonas nitroreducens</span></i><span style="font-family:Verdana;"> (PnGGT). Using recombinant PnGGT, we have shown previously that Trp385, Phe417, and Trp525 are key amino acid residues for recognition of acceptor substrates at the PnGGT active site. Here, we demonstrate that a recombinant W525D mutant of PnGGT produces L-theanine from ethylamine and L-glutamine more efficiently than wild-type PnGGT, attributable to an increased ratio of transfer activity to hydrolysis activity. An efficient production of L-theanine was achieved by immobilizing </span><i><span style="font-family:Verdana;">Escherichia coli</span></i><span style="font-family:Verdana;"> cells expressing the W525D PnGGT mutant (</span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D) using 2% alginate as the supporting material. The highest L-theanine production using immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana;">-W525D, representing a conversion rate of 90%, w</span><span style="font-family:Verdana;">as</span><span style="font-family:Verdana;"> achieved in optimal reaction conditions of pH 10, 40<span style="white-space:nowrap;">°</span></span><span style="font-family:;" "=""><span><span style="font-family:Verdana;">C, and a substrate molar ratio of L-glutamine to ethylamine of 1:10. The immobilized </span><i><span style="font-family:Verdana;">E. coli</span></i><span style="font-family:Verdana
关 键 词:L-THEANINE Γ-GLUTAMYLTRANSPEPTIDASE Pseudomonas nitroreducens Escherichia coli Immobilization
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