A kind of synthetic method of vitamin A acetate
A synthesis method and acetate technology, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of high energy consumption, cumbersome operation steps, high operation risks, etc., and achieve mild reaction conditions and products. The effect of high purity and high reaction yield
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[0037] According to a preferred embodiment of the present invention, the dehydratase is obtained according to the following method: the enzyme gene is obtained from the genus or strain by cloning or chemical synthesis, and the enzyme gene is introduced into a prokaryotic expression vector to obtain a recombinant vector Afterwards, the recombinant vector is transformed into Escherichia coli to obtain a recombinant strain, and the recombinant strain is fermented, isolated and purified to obtain the dehydratase; the gene sequence of the enzyme gene is SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6. Among them, the methods of obtaining the enzyme gene by cloning or chemical synthesis, introducing the enzyme gene into a prokaryotic expression vector to obtain a recombinant vector, and transforming the recombinant vector into Escherichia coli to obtain a recombinant strain are all well known to those skilled in the art, and will not be repeat...
preparation example 1
[0067] This preparation example is used to illustrate the synthesis of the dehydratase provided by the present invention.
[0068] (1) Enzyme synthesis: the enzyme gene of Chryseobacterium meningosepticum (obtained by chemical synthesis of Sangon Bioengineering (Shanghai) Co., Ltd., the gene sequence of the enzyme gene is SEQ ID NO: 4) was cloned into the pET25 vector , and then transform the resulting recombinant vector into Escherichia coli BL21 (DE3) to obtain a recombinant strain, which was shaken in TB medium containing 100 μg / mL ampicillin at 30°C and 180 rpm until the OD600 value was 0.6-0.8 , and then add the inducer IPTG to the bacterial solution to a final concentration of 1 mM, and continue to cultivate for 24 hours. The bacterial liquid was centrifuged at 4°C and 6000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected. The collected cells were resuspended in Tris-HCl buffer (20mM Tris-HCl, 300mM NaCl, pH 8.0), ultrasonically disr...
preparation example 2
[0071] This preparation example is used to illustrate the synthesis of the dehydratase provided by the present invention.
[0072] (1) Enzyme synthesis: the enzyme gene of Stenotrophomonas maltophilia (obtained by chemical synthesis of Sangon Bioengineering (Shanghai) Co., Ltd., the gene sequence of the enzyme gene is SEQ ID NO: 1) was cloned into pET25 Then transform the obtained recombinant vector into Escherichia coli BL21(DE3) to obtain a recombinant strain, which was shaken in TB medium containing 50 μg / mL ampicillin at 25°C and 100 rpm until the OD600 value was 0.6 -0.8, then add the inducer IPTG to the bacterial solution to a final concentration of 1.2mM, and continue to cultivate for 30h. The bacterial liquid was centrifuged at 4°C and 7000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected. The collected cells were resuspended in Tris-HCl buffer (20mM Tris-HCl, 300mM NaCl, pH 8.0), ultrasonically disrupted, centrifuged at 4°C, 7000r...
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