Artificial biocatalyst for converting fatty acid into olefin by using hydrogen peroxide
A technology of fatty acid decarboxylase and olefin, which is applied in the field of protein engineering, can solve the problems of complex reaction system, limited application prospect, narrow substrate range, etc., and achieve the effect of simple reaction system, wide substrate range and low cost
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Embodiment 1P450
[0034] The construction of embodiment 1P450BSβ gene expression vector
[0035] The P450BSβ fragment synthesized from the whole gene (sequence shown in SEQ ID NO: 3, synthesized by Suzhou Jinweizhi Biological Co., Ltd.) was double-digested with restriction endonucleases NcoI and XhoI (New England Biolabs) according to the instructions , T4 ligase (New England Biolabs) was used to connect to the expression vector pET28a cut by NcoI and XhoI. The ligation product was transformed into Escherichia coli DH5α competent cells (Tiangen Biochemical Technology Co., Ltd.). Pick the successfully transformed monoclonal colony from the solid LB medium plate containing 50 μg / ml kanamycin, and place it in the LB liquid medium containing the same concentration of kanamycin at 37°C with a shaker speed of 220rpm / min conditions overnight. The recombinant plasmid was extracted from the cultured overnight bacterial solution using a small plasmid extraction kit (Tiangen Biochemical Technology Co., ...
Embodiment 2P450
[0036] Large amount of expression and purification of embodiment 2P450BSβ mutant protein
[0037] Pick glycerol bacteria stored in a -80°C ultra-low temperature refrigerator and inoculate into 5ml TB liquid medium
[0038] (Kan / Cam) cultured overnight at 37°C. Inoculate 500ml of TB culture medium (Kan / Cam) with the bacterial solution cultivated overnight, and carry out expansion cultivation at 37°C with a shaker speed of 220rpm / min; 600 At about 0.6, add precursor ALA (δ-aminolevulinic acid) at a final concentration of 0.5 mM and 1 mM IPTG to induce expression, and culture at 22° C. with a shaker speed of 220 rpm / min for 16 h. Centrifuge at 5000rpm / min for 10 min, collect the bacteria, and resuspend the bacteria with 50ml of buffer A (0.1M KPi, 0.3M KCl, 20% glycerol, pH 7.0), and then use ultrasound to disrupt. Centrifuge at 10000 rpm / min at 4°C for 45 min to obtain the supernatant. Put the supernatant containing the target protein into the prepacked nickel ion affinity co...
Embodiment 3P450
[0039] Embodiment 3P450BSβ mutant hydrogen peroxide reaction system
[0040] Reaction system for oxidative decarboxylation with P450BSβ mutant. The decarboxylation reaction system contained buffer A, 2.5 mM of purified P450BSβ mutant, 2.5 mM of fatty acid substrates with different chain lengths, 5 mM of hydrogen peroxide, and the final volume was 1 ml. The reaction was carried out at 10° C., pH 7.0, and 120 rpm / min for 30 min. The results are shown in Table 1 below. P450BSβ mutant protein can not only catalyze C 6 -C 18 Straight-chain fatty acids with different chain lengths can also catalyze naturally occurring unsaturated long-chain fatty acids such as oleic acid and linoleic acid, successfully expanding the range of catalyzed substrates. The application prospect of the reaction system in the field of synthesis and the field of energy is broadened.
[0041] Table 1 Hydrogen peroxide reaction system for catalytic decarboxylation of P450BSβ mutant under different substrat...
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