Supplementary strategy for improving catalytic synthesis of L-theanine from gamma-glutamyl transpeptidase
A technology of glutamyl transpeptidase and glutamyl transpeptidase enzyme solution, which is applied in the direction of acyltransferase, transferase, enzyme, etc., can solve the problems of low L-theanine production, low pH, L-glutamine Solve problems such as low solubility of amides, achieve high conversion rate and high application value
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Embodiment 1
[0020] Embodiment 1: fermenter culture of recombinant Bacillus subtilis and concentration of crude enzyme solution
[0021] After culturing the recombinant Bacillus subtilis B.subtilis / pMA5-ggt in the seed medium for 16 hours, inoculate it in a fermenter containing 1.8L fermentation broth at an inoculum size of 6-10% (volume ratio), centrifuge after cultivating for 48 hours Obtain the supernatant enzyme solution, add 70% ammonium sulfate for precipitation, dialyze the dialysis bag overnight to desalt, dissolve in 50mM Tris-HCl buffer solution (pH 8.0), and obtain the enzyme concentrated solution.
Embodiment 2
[0022] Example 2: Preliminary identification of the ability of concentrated enzyme solution (BsGGT) to synthesize theanine
[0023] The smallest conversion system is: 10mL of reaction liquid contains: glutamine (20mM), ethylamine (20mM), GGT (60U / ml), the mixed system without enzyme solution is used as a control, and the reaction solution is added after catalyzing the reaction for 5 hours at 37°C The same volume of 10% trichloroethylamine was used to terminate the reaction. The resulting reaction mixture was filtered through a membrane (0.45 [mu]m; Millipore) for HPLC analysis. Simultaneous mass spectrometric analysis of the produced theanine
Embodiment 3
[0024] Example 3: Further optimization of the enzyme catalytic system by substrate flow-adding strategy
[0025] Get concentrated enzyme solution, enzyme content 60U / mL, add substrate glutamine and ethylamine therein every two hours, the addition amount of glutamine is fixed at 20mM, and donor acceptor ratio is at (1:4 -1:12) fluctuations to find the most suitable substrate ratio, sample analysis before each substrate addition, adjust the pH to 10 with diluted HCl after addition, and terminate the reaction when the production of theanine no longer increases.
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