Method for preparing L-tertiary leucine by immobilized coupled bi-enzyme
A technology of tert-leucine and leucine dehydrogenase, which is applied in the field of immobilized coupling double enzymes to prepare L-tert-leucine, which can solve the problems of low theoretical yield, low utilization efficiency, and large amount of enzyme , to achieve high product conversion rate, improve utilization efficiency, and mild reaction conditions
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Embodiment 1
[0046] A method for preparing L-tert-leucine by immobilizing coupled double enzymes, the reaction principle is as follows: figure 1 As shown, it specifically includes the following steps:
[0047] (1) Preparation of cellulose carrier: Take 0.2g~10g microcrystalline cellulose, add 0.5~10mL deionized water to dissolve, slowly add pre-cooled phosphoric acid until the final concentration of phosphoric acid is 20%~85%, and ice bath for 0.5h~ Stir for 5 hours, then add 20-200 mL of ice water, centrifuge to discard the supernatant, wash with ice water, and finally adjust the pH to neutral to obtain the cellulose carrier.
[0048] (2) Preparation of immobilized coupled double enzymes: specifically include:
[0049] 1) Construct the E.coli engineering bacteria of the leucine dehydrogenase gene marked by the docking module. The sequence of the leucine dehydrogenase gene marked by the docking module is shown in SEQ ID 1. The steps are as follows:
[0050] a. Using the plasmid pUC18-ldh...
Embodiment 2
[0066] Step (1) to step (2) are the same as embodiment 1, and step (3) is as follows:
[0067] Use 5% ammonia water to prepare 130g / L of trimethylpyruvate and 95g / L of ammonium formate, and make the pH of the mother solution twice as high as about 9; use deionized water to prepare 4mM NADH; in 1mL reaction system, Add 0.5mL mother liquor, 0.05mL 4mM NADH, add 0.45mL immobilized coupled enzyme with a total protein concentration of 0.38mg / mL, react at 25°C, and shake intermittently. Samples were taken at 0.5, 1, 2, 3, 4, 5, 8 and 24 hours, then diluted 5 times, bathed in boiling water for 15 minutes, and centrifuged to obtain the supernatant for high performance liquid chromatography analysis.
[0068] The analysis conditions are: standard L-tert-leucine, D-tert-leucine and product L-tert-leucine are all analyzed by Agilent 1200 high performance liquid chromatography, Chirex 3126 chiral chromatographic column, mobile phase or elution phase It is 2mM copper sulfate, 5% isopropan...
Embodiment 3
[0072] Step (1) and step (2) are the same as embodiment 1, and step (3) is as follows:
[0073] Use 5% ammonia water to prepare 182g / L of trimethylpyruvate and 95g / L of ammonium formate to make the pH of the 2-fold mother solution about 9; use deionized water to prepare 4mM NADH; in 1mL reaction system, Add 0.5mL mother liquor, 0.05mL 4mM NADH, add 0.36mL immobilized coupled enzyme with a total protein concentration of 3mg / mL, react at 25°C, and shake continuously. Samples were taken at 2, 5, 8, 11 and 24 hours, then diluted 10 times, bathed in boiling water for 15 minutes, and centrifuged to take the supernatant for high performance liquid chromatography analysis.
[0074] Analysis condition is as embodiment 2, and result shows that product concentration and reaction time are in linear relationship (through linear regression analysis R 2 =0.9979), indicating that under the condition of high substrate concentration, the coupled dizyme will not produce substrate inhibition. 2...
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