Tyrosine phenol lyase engineering bacteria, construction method and applications thereof
A technology of tyrosine phenol and construction method, which is applied in the field of genetic engineering, can solve the problems of low yield of L-DOPA, difficulty in controlling reaction conditions, and many by-products, and achieve large soluble expression, low cost, and high yield Effect
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Embodiment 1
[0030] 1. Prepare BL21 (DE3) as competent cells
[0031] Use the TAKARA Competent Cell Preparation Kit and operate according to the instructions to prepare BL21 (DE3) competent cells.
[0032] 2. Whole gene synthesis of tyrosine phenol lyase gene fragment
[0033] According to the sequence provided by Gene ID: X66978.1, the whole gene fragment of tyrosine phenol lyase was synthesized.
[0034] 3. Construction of tyrosinase expression plasmid pMBP-TPL
[0035] The whole gene fragment of tyrosinase was subcloned to the downstream of the maltose binding protein gene fragment on pSYNO-1 plasmid.
[0036] 4. The expression plasmid pMBP-TPL is introduced into competent cells
[0037] 1) Immediately insert the competent state into an ice-water bath for 3 minutes after taking it out from -70°C;
[0038] 2) Add 1 microliter of plasmid pMBP-TPL to the competent state in the ultra-clean bench, flick and mix well, immediately insert into the ice water bath for 25 minutes, and let stan...
example 2
[0043] tyrosine phenol lyase
[0044] LB medium: tryptone 10g / L, yeast extract 0.5 g / L, sodium chloride 10g / L, pure water.
[0045] Fermentation medium: tryptone 12g / L, yeast extract 24g / L, glycerol 5g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate trihydrate 16.43 g / L, pure water.
[0046] 1) Pick a single colony and inoculate it into a 4ml LB medium test tube, add kanamycin (50mg / L), culture at 37°C, 220rpm for 12h, and obtain first-grade seeds;
[0047]2) The primary seeds were inoculated into 100ml of fermentation medium shake flask, 37°C, 220rpm, cultured for 4h, added IPTG to a final concentration of 1mM, 25°C, 220rpm, cultured for 12h;
[0048] 3) Centrifuge the bacterial liquid in step (2) to collect the bacterial cells, and place in a -20°C refrigerator.
example 3
[0049] Example 3: Conversion of tyrosine phenol lyase to produce L-dopa
[0050] 1) 1L substrate solution: 14g / L sodium pyruvate, 10g / L catechol, 40g / L ammonium chloride, 2g / L sodium sulfite, 1g / L EDTA, adjust pH to 8.0;
[0051] 2) To 35g of bacterial cells, add 1L of substrate solution, stir well, seal and shake at 25°C;
[0052] 3) Add the substrate (equal amount of sodium pyruvate and quinone) every half hour, and control the concentration of the two substrates to not be higher than 10g / L;
[0053] 4) When the residual concentration of catechol falls below 1g / L, stop the reaction, and the concentration of L-dopa accumulates to above 100g / L.
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