Beta-dehalogenase gene and preparation method of 3-hydracrylic acid
A hydroxypropionic acid and dehalogenase technology, applied in microorganism-based methods, biochemical equipment and methods, enzymes, etc., can solve difficult large-scale industrial production, many by-products of 3-hydroxypropionic acid, separation and purification Difficulties and other problems, to achieve the effect of high conversion rate, large equipment investment, and high technical difficulty
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Embodiment 1
[0046] Embodiment 1 has the construction of the recombinant plasmid of β-dehalogenase gene (referring to figure 1 )
[0047] (1) Construction and screening of gene library
[0048]Extract the genome of Bacillus sp. 3-CPA15 with the preservation number CGMCC No.4196, take 10 μl of it and digest it with 0.001U-0.1U of Sau3A at 37°C for 0.5 hours, and perform agarose gel on the digested fragment Electrophoresis, select the enzyme concentration of 1Kb-10Kb for the cleavage fragment, prepare the genome cleavage fragment under the same reaction conditions, and obtain the cleavage product a. The pUC19 vector was digested with BamHI overnight at 37°C to obtain the digested product b. Mix the digestion products a and b, add T4 ligase, and ligate overnight at 16°C. Get connection system c. The connection system c was transformed into E.coli XL-Blue super competent cells, and coated with 100 μg / ml ampicillin, 0.5 mmol / L IPTG (Isopropylβ-D-1-Thiogalactopyranoside) and 40 μl X-Gal (5-B...
Embodiment 2
[0055] Example 2 Construction of recombinant E.coli BL21(DE3)-CondonPlus producing β-dehalogenase.
[0056] (1) Induced expression of recombinase
[0057] The recombinant cells carrying the pBHD plasmid were inoculated in 5 ml LB liquid medium (containing 50 μg / ml kanamycin and 34 μg / ml chloramphenicol), and cultured overnight at 37° C. and 220 rpm. Inoculate the above culture into a 500ml Erlenmeyer flask containing 100ml LB medium (containing 50μg / ml kanamycin and 34μg / ml chloramphenicol) at a ratio of 2:100, and cultivate to OD at 37°C and 220rpm 600 =0.6-0.8, add IPTG with a concentration of 0.5mol / L to a final concentration of IPTG of 0.5mmol / L. Under the condition of 30°C, culture was induced on a shaker at 220rpm for 6h.
[0058] The cells were collected by centrifugation and washed twice with pre-cooled loading buffer (20mmol / L sodium phosphate buffer, 500mM NaCl, 20mM imidazole, pH 7.5). The cells were resuspended in the above buffer at a ratio of 10 ml buffer / g we...
Embodiment 3
[0064] Get 4ml of enzyme solution A in Example 2, divide into four parts, each part of 1ml, add 3-chloropropionic acid solution respectively, so that the molar ratio of 3-chloropropionic acid relative to the total amount of 3-chloropropionic acid solution and pure enzyme solution The concentration is 10mmol / L. The four solutions were placed in a constant temperature shaker with a temperature of 20°C, 30°C, 40°C and 50°C and a rotation speed of 180rpm. After 24 hours, the substrate 3-chloropropionic acid and the product 3 were detected by liquid phase detection. - the concentration of hydroxypropionic acid, its retention time on the liquid phase as Figure 6 As shown, the peak on the left is 3-hydroxypropionic acid (13.12min), and the peak on the right is 3-chloropropionic acid (20.73min). The conversion rate of the substrate 3-chloropropionic acid is calculated, and the results are shown in Table 1. From the data in Table 1, it can be seen that the conversion rate increases w...
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