Application of recombinant Bacillus subtilis γ-glutamyltranspeptidase to the production of l-theanine |
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Institution: | 1. Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 600, Taiwan;2. Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy;3. Istituto di Biostrutture e Bioimmagini, CNR, Napoli, Italy;1. Department of Chemical Sciences, University of Naples Federico II, Via Cintia, Naples I-80126, Italy;2. Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone, 16, Naples 80133, Italy;3. Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan |
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Abstract: | l-Theanine, which has seen increasing use in the functional food industry, can be prepared via enzymatic synthesis using γ-glutamyltranspeptidase (GGT; EC 2.3.2.2). In this study, the GGT from Bacillus subtilis 168 was cloned and expressed as a secreted protein using Escherichia coli BL21(DE3). The enzymatic properties of the GGT and the optimal conditions for the enzymatic synthesis of l-theanine were investigated in detail. The activity of the enzyme was optimal at pH 10; the optimal temperature was 50 °C. Desirable pH stability was observed between pH 5 and pH 12, and adequate thermostability was seen at 50 °C. In 5 h at 37 °C, the enzyme converted 200 mM l-glutamine and 2.2 M ethylamine to l-theanine with a final yield of 78%. Yields of l-theanine decreased to 58% when using 500 mM Gln and 45% when using 1 M Gln. The yield of l-theanine obtained at high substrate concentration provides the basis for the industrial-scale production of l-theanine. |
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Keywords: | γ-Glutamyltranspeptidase (GGT) Industrial-scale production Enzymatic synthesis |
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