L-theanine enzymatic transformation preparation method
A technology of theanine enzymatic method and glutamic acid, which is applied in the field of enzymatic conversion and preparation of L-theanine, achieves the effects of high substrate conversion rate, mild reaction conditions, and reduced production costs
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Embodiment 1
[0032] L-theanine enzymatic transformation preparation method, its specific steps are as follows:
[0033] 1. Add 21 g of Bacillus subtilis NX-2 cells obtained by centrifuging 1000 mL of fermentation broth into 1000 mL of transformation liquid, which contains 20 g of L-glutamic acid-γ-methyl ester, 45 g of ethylamine, and 10 mL of 5% Tween- 80, 10mL of 1mol / L potassium chloride, 30% hydrochloric acid to adjust the pH to 10, and react at 35°C for 16h. The concentration (g / mL) of L-theanine in the reaction solution was 1.64%, and the molar conversion rate of the substrate L-glutamic acid-γ-methyl ester reached 76%.
[0034] 2. Centrifuge the reaction solution to remove bacteria, adjust the pH of the supernatant to 3 with 30% hydrochloric acid, heat up the acidification solution to 70-80°C, add activated carbon for decolorization, and pass the decolorization solution through a 732-type cation exchange resin column for adsorption. Elute the L-theanine saturated adsorption column ...
Embodiment 2
[0036] L-theanine enzymatic transformation preparation method, its specific steps are as follows:
[0037] 1. Add 18 g of Bacillus subtilis SK11.004 cells obtained by centrifuging 1000 mL of fermentation broth into 1000 mL of transformation liquid, which contains 40 g of L-glutamic acid-γ-ethyl ester, 80 g of ethylamine, and 10 mL of 1% hexadecane Trimethylammonium bromide, 10mL 1mol / L sodium chloride, 30% hydrochloric acid to adjust the pH to 10, and react at 35°C for 20h. The concentration (g / mL) of L-theanine in the reaction solution was 3.10%, and the molar conversion rate of the substrate L-glutamic acid-γ-ethyl ester reached 78%.
[0038]2. Centrifuge the reaction solution to remove bacteria, adjust the pH of the supernatant to 3 with 30% hydrochloric acid, heat up the acidification solution to 70-80°C, add activated carbon for decolorization, and pass the decolorization solution through a 732-type cation exchange resin column for adsorption. Elute the L-theanine satura...
Embodiment 3
[0040] L-theanine enzymatic transformation preparation method, its specific steps are as follows:
[0041] 1. Add 15 g of Escherichia coli SH642 (containing recombinant plasmid pUC18-GGT) cells obtained by centrifugation of 1000 mL of fermentation broth into 1000 mL of transformation liquid, which contains 60 g of L-glutamic acid-γ-propyl ester, 112 g of ethylamine, 20 mL 1% polyethylene glycol octylphenyl ether, 10mL 1mol / L calcium chloride, 30% hydrochloric acid to adjust the pH to 10, and react at 35°C for 26h. The concentration (g / mL) of L-theanine in the reaction solution was 3.98%, and the molar conversion rate of the substrate L-glutamic acid-γ-propyl ester reached 72%.
[0042] 2. Centrifuge the reaction solution to remove bacteria, adjust the pH of the supernatant to 3 with 30% hydrochloric acid, heat up the acidification solution to 70-80°C, add activated carbon for decolorization, and pass the decolorization solution through a 732-type cation exchange resin column f...
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