Function of novel R-selective styrene monooxygenase
A monooxygenase, styrene technology, applied in oxidoreductase, biochemical equipment and methods, applications, etc., can solve the problems of low enzyme catalytic efficiency, narrow substrate spectrum, etc., to achieve high activity, wide substrate spectrum effect
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Embodiment 1
[0016] Example 1 Styrene monooxygenase SeStyA co-expression vector construction and activity verification
[0017] pETAB is a plasmid constructed in our previous work (J.Mol.Catal.B:Enzym.2010,67:236-241), which contains DNA fragments of StyA, linker and StyB. The present invention replaces the StyA gene in pETAB with SeStyA, retains the linker and StyB, thereby constructing an artificial two-component plasmid pETSeStyAB with a complete catalytic system.
[0018] The specific method is as follows:
[0019] (1) Using the plasmid pETAB as a template, the Linker region of pETAB was introduced into the Spe I restriction site by site-directed mutagenesis. Primer used 5'-GCCGACCATTGCAGCCTGA ACTAGT CTCCGCTGGCCATGCCAGC-3' and 5'-CTGGCATGGCCAGCGGAG ACTAGT TCAGGCTGCAATGGTCGG-3'. The resulting plasmid was double-digested with Nde I and Spe I to obtain the digested vector fragment. The specific method is a conventional method in the art.
[0020] (2) Amplify the DNA fragment of Se...
Embodiment 2
[0022] The biocatalysis of embodiment 2 different substrates
[0023] The whole cell reaction system was the same as in Example 1, and the substrate concentration was 4 mM. The substrate spectrum is shown in Table 1, as can be seen from Table 1, SeStyA has higher catalytic activity to styrene, 1,2-dihydronaphthalene, 3-bromostyrene, and wherein to styrene and 1,2- The conversion rate of dihydronaphthalene can reach 100%, but the activity of 2-bromostyrene, 4-bromostyrene and 4-chlorostyrene is low; SeStyA catalyzes 4-bromostyrene and 4-chlorostyrene to generate S Type epoxy products, while other substrates are catalyzed to generate R-type epoxy products.
[0024] Table 1 SeStyA whole cell biotransformation results
[0025]
[0026]
[0027] a represents the reaction time of 4 hours.
Embodiment 3
[0028] The enzyme kinetic parameter determination of embodiment 3SeStyA
[0029] Heterologous expression of SeStyA: transfer the pETSeStyA plasmid into E.coli BL21(DE3) by chemical method, coat the LB plate containing kanamycin (50 μg / mL), and culture overnight at 37°C. Pick a single clone into LB medium containing kanamycin (50 μg / mL), and culture overnight at 37° C. and 180 rpm. Inoculate in TB medium with 1% inoculum amount, culture with shaking at 37°C for 3 hours, wait until OD 600At about 0.8, add IPTG with a final concentration of 0.05mM, adjust the temperature to 20°C and induce for 18h, then centrifuge at 6000rpm and 4°C for 10min, discard the supernatant, wash the bacteria twice with normal saline, and add 0.1M pH 7.0 The cells were resuspended in potassium phosphate buffer and mixed evenly, the cells were disrupted by ultrasonic waves (working conditions: power 200W, working for 3s, intermittent for 3s, working times 99 times), centrifuged, and the supernatant was ...
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