Biocatalytic synthesis method of piceatannol
A biocatalysis and whitening technology, applied in biochemical equipment and methods, microbial-based methods, botany equipment and methods, etc., can solve the problems of conversion rate decline, achieve high conversion rate, excellent synthesis effect, and increase yield Effect
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Embodiment 1
[0035] The construction of embodiment 1 carrier
[0036] A. First, artificially synthesize the HpaB enzyme gene. The sequence is shown in SEQ ID No.1. Cloning site: BamHIHindIII is cloned into the first multiple cloning site of the pETduet-1 vector to obtain pETduet-HpaB, and then artificially synthesized The HpaC enzyme gene, whose sequence is shown in SEQ ID No.2, was further cloned into the second multiple cloning site of the above-mentioned pETduet-HpaB vector through the cloning site: NdeI XhoI, to obtain the pETduet-HpaBC (MCS) vector.
[0037] The artificially synthesized HpaC2 enzyme gene, whose sequence is shown in SEQ ID No.3, was cloned into the pETduet vector through the cloning site: NdeI XhoI, to obtain the pETduet-HpaC2 vector.
[0038] The HpaB enzyme gene sequence is shown in SEQ ID No.1, and the HpaB enzyme is derived from P.aeruginosa strain;
[0039] The HpaC enzyme gene sequence is shown in SEQ ID No.2, denoted as HpaC1, and the HpaC enzyme is derived fro...
Embodiment 2
[0078] The construction of embodiment 2 recombinant bacteria
[0079] Construct recombinant bacteria with the above expression vector
[0080] Construction of recombinant bacteria:
[0081] Take the pACYCDuet-HpaBC2 plasmid to construct recombinant bacteria as an example:
[0082] The recombinant plasmid pACYCDuet-HpaBC2 was transformed into Escherichia coli BL21(DE3) and cultured in LB solid medium containing chloramphenicol (50ug / ml) to obtain the recombinant cell E.coli pACYCDuet-HpaBC2.
[0083]For other expression methods such as vector pETduet-HpaBC (MCS) plasmid constructing recombinant bacteria, refer to the above.
[0084] Plasmid (pRSFduet-HpaC2) and pETDuet-HpaB are co-transfected into competent cells to obtain pETDuet-HpaB+pRSFDuet-HpaC2 recombinant bacteria;
[0085] The protein expression of each recombinant bacteria:
[0086] The constructed Escherichia coli recombinant bacteria were inoculated in 3 mL of LB liquid medium (peptone 10 g / L, yeast powder 5 g / L,...
Embodiment 3
[0087] Example 3 Biotransformation of Resveratrol to Pixanol
[0088] Fermentation is carried out with the above-mentioned constructed recombinant bacteria to obtain the bacterial liquid, and then the reaction is carried out. Add the corresponding concentration of bacterial solution in the reaction bottle, OD 600 For 10-32, the substrate resveratrol was dissolved in 2-hydroxypropyl-β-cyclodextrin, 1% Tween 80 was added, and 200 mM phosphate buffer was used, in which 10% (v / v ) of glycerin. 30° C., 200 rpm to start the reaction. Samples were taken at the corresponding time, 50 μL of the sample was taken, 950 μL of acetonitrile was added, shaken, centrifuged, the supernatant was taken to pass through the membrane, and analyzed by HPLC.
[0089] The results showed that at 20mM and 30mM substrate concentrations, pETDuet-HpaB+pRSFDuet-HpaC2, pACYCDuet-HpaBC2(rbs) had excellent effects, and pACYCDuet-HpaBC2(rbs) had the best effect, as Figure 4 Shown: the most product was obtai...
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