A method for preparing 3-phenyl-l-serine or derivatives thereof and ethyl ester thereof
A technology for serine and derivatives, which is applied in the field of preparation of chiral 3-phenyl-L-serine or its derivatives and ethyl esters thereof, can solve the problems of poor stereoselectivity, low conversion rate, difficulty in product separation and purification, etc.
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Embodiment 1
[0111] Example 1: Gene cloning and expression vector construction
[0112] Four wild-type L-threonine transaldolases, namely: YH1058 (Suzhou Pilot Biotechnology Co., Ltd., amino acid sequence as shown in SEQ ID NO.12), and LTTA sp01 (amino acid sequence as shown in SEQ ID NO.13) The gene sequences of LTTA sp02 (amino acid sequence shown in SEQ ID NO.14) and LTTA sp03 (amino acid sequence shown in SEQ ID NO.15) can be obtained in the NCBI database, and can be obtained through gene synthesis and molecular cloning. The pET30a expression plasmid containing the threonine transaldolase gene was constructed. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) cells to obtain recombinant bacteria.
Embodiment 2
[0113] Example 2: Expression of recombinant L-threonine transaldolase
[0114] The recombinant bacteria in Example 1 were inoculated into LB medium (10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, pH 7.0) containing 50 μg / mL kanamycin, and cultured at 37°C overnight. The overnight culture was transferred to TB medium (peptone 12g / L, yeast extract 24g / L, glycerol 4mL / L, potassium dihydrogen phosphate 2.31g / L, dipotassium hydrogen phosphate 12.54g / L), and cultured at 37°C until OD 600 To 0.6-0.8, IPTG was added at a final concentration of 0.4 mM, and expression was induced at 30°C overnight. After the cells were collected by centrifugation, they were resuspended in 20 mM phosphate buffer, pH 7.0, 4 times the wet weight of the cells. The cells were disrupted by sonication, centrifuged, and the supernatant was taken for reaction or frozen at -20°C.
Embodiment 3
[0115] Example 3: Testing of wild-type L-threonine transaldolase activity at low substrate concentrations - no acetaldehyde removal system
[0116] The final concentration of 5g / L p-methylsulfonylbenzaldehyde, 6.4g / L L-threonine, 1g / L magnesium chloride, 0.005g / L pyridoxal phosphate, 100mM phosphate buffer (pH7. 5) Heating to 30°C with magnetic stirring, adding 20 μl of L-threonine transaldolase (YH1058 (Suzhou Pilot Biotechnology Co., Ltd.) obtained in Example 2 at a concentration of 30 g / L, as well as LTTA sp01, LTTA sp02 and LTTA sp03) enzyme solution, start stirring reaction, 16 hours later sampling HPLC detection, the detection results are shown in Table 5.
[0117] Table 5 Wild-type L-threonine transaldolase activity detection results
[0118] Numbering serial number Conversion rate(%) Enantiomeric excess (ee%) a
Diastereomeric Ratio (DR) b
YH1058 SEQ ID NO. 12 93.6 98.6 94.7:5.3 LTTA sp01 SEQ ID NO. 13 91.5 98.5 94.2:5.8 ...
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