Mn2+ enhances theanine-forming activity of recombinant glutamine synthetase from Bacillus subtilis in Escherichia coli

Abstract

Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0-100 mM) of Mn 2+ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9-A (details are described in ''Materials and methods'') and 0.1% (w/v) lactose were selected as culture medium and inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the specific activity of the recombinant GS is 6.2 U/ mg which is approximate to that (6.4 U/mg) induced in LB in the presence of 10 mM Mn 2+ at optimal pH 7.5. The activity is markedly higher activated by Mn 2+ than that by other nine bivalent cations. Furthermore, M9-B (5 lM Mn 2+ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and 13.1 g/L by paper chromatography and HPLC, respectively.

From Tea Leaves to Factories: A Review of Research Progress in l-Theanine Biosynthesis and Production

“…75 The recombinant GS exhibited the same properties and 30-fold higher enzyme productivity compared to that of original P. taetrolens Y-30. 75 In addition, the GSs from Bacillus subtilis, 76 Pseudomonas fluorescens, 77 and Bacillus megaterium 78 were also investigated and characterized to possess the capability of synthesizing Ltheanine.…”

“…taetrolens Y-30 . In addition, the GSs from Bacillus subtilis, Pseudomonas fluorescens, and Bacillus megaterium were also investigated and characterized to possess the capability of synthesizing l -theanine.…”

L-Theanine: properties, synthesis and isolation from tea

“…The enzyme glutamine synthetase and related enzymes, originating from Escherichia coli ,60 Pseudomonas taetrolens ,61 Methylovorus mays 62 and Bacillus subtilis ,63 have been utilised for the synthesis of theanine from glutamic acid and ethylamine. Unlike the glutaminase biosynthetic pathway discussed previously, this glutamic acid transformation, while using a cheaper and more stable starting material, requires a continuous supply of ATP to drive the reaction 62.…”

Studies on the Biochemical Formation Pathway of the Amino Acid l-Theanine in Tea (Camellia sinensis) and Other Plants

“…Wu et al identified 20 CsGS genes, including several unconfirmed CsTSs . Studies on bacteria, C. sinensis , and pea seeds have shown that GS can convert Glu and free ammonia to Gln, and also transfer Glu to l -theanine in the presence of ethylamine. ,− These findings suggest that GS and TS are very similar not only in their DNA sequences, but also in their enzymatic activities. As GS/TS are present in many plants, it is still unknown why l -theanine accumulates only in C. sinensis , and barely or not at all in other plant species.…”