Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 67, Issue 51, Pages 14129-14136Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.9b06140
Keywords
gamma-glutamyltranspeptidase; Bacillus subtilis; optimization; synthesis of l-theanine
Funding
- National Key Research and Development Program of China [2018YFD0400600, 2018YFD0400400]
- Key Scientific and Technological Project of Anhui Province of China [17030701014, 18030701146]
- Open Fund of State Key Laboratory of Tea Plant Biology and Utilization [SKLTOF20180107]
- China Postdoctoral Science Foundation [2019M651013]
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In this study, the gene of gamma-glutamyltranspeptidase (GGT) from Bacillus amyloliquefaciens (BaGGT) controlled by the P-lac promoter was cloned into Bacillus subtilis to construct two recombinant vectors with either one or two signal peptides to drive extracellular secretion. After optimization; 90 +/- 0.2 mg/L BaGGT was obtained when the inducing conditions were 24 h and 80 mu M (IPTG). The properties of BaGGT were measured, showing that the optimal reaction conditions were 40 degrees C and pH 9.0 with 55.0 +/- 0.5 U/mg enzymatic activity. K-m and V-max were 0.214 mM and 88.13 mu mol/min/mg. BaGGT could be stored for 72 h with 90% of the initial activity at 40 degrees C and retained more than 50% of the initial activity after being maintained at different pH values for 24 h. Finally, enzymatic synthesis of L-theanine was performed with the optimal conditions: 20 mM L-Gln, 100 mM ethylamine HCl, 0.5 U/mL BaGGT, incubated at 40 degrees C for 6 h, 200 rpm.
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