A kind of epoxide hydrolase and its coding gene and application
A technology of epoxide and hydrolase, which is applied in the field of enzyme catalysis and can solve problems such as inability to prepare
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Embodiment 1
[0023] Example 1: Cloning Containing the Epoxyhydrolase Gene
[0024] Purchase standard strain freeze-drying tubes numbered DSM20162 from DSMZ, Germany.
[0025] 1LTsukamurella paurometabola culture medium is as follows: Trypticase Soy Broth (BBL11768, Oxoid CM129 or Merck 5459) 30.0g; distilled water 1000ml; pH7.3. Sterilize at 121°C for 15min. Cultivate in a constant temperature shaker at 200rpm at 28°C for 1 to 3 days, collect the bacteria by centrifugation, and extract the genome of Tsukamurella paurometabola according to the instructions of the Genome Extraction Kit of Jerry Company, and amplify it with the following two primers:
[0026] F: 5'-CGC GGATCC ATGACGATCACCCCGCACACCGTG-3' (SEQ ID NO: 3);
[0027] R: 5'-CCC AAGCTT TCAGCCCGCGGGGTGGTTTCTCC-3' (SEQ ID NO: 4);
[0028] The full length of the epoxyhydrolase gene was obtained.
[0029] Toyobo KOD enzyme PCR reaction system is: H 2 O 32.5ul, buffer 5ul, 2mm dNTP5ul, MgCl 2 2.5ul, template 2ul, 20mM F / R primer ...
Embodiment 2
[0030] Example 2: Obtaining whole cells containing epoxyhydrolase
[0031]The obtained transformants were inoculated into LB liquid medium containing 50 μg / ml kanamycin resistance, cultured at 37° C. for 12 h, and then inoculated into fresh 50 μg / ml kanamycin containing 50 μg / ml kanamycin In the mycin-resistant LB liquid medium, cultivate at 37°C until the bacterial cell concentration OD600 is about 0.6, then add IPTG with a final concentration of 0.1mM to the LB liquid medium, induce culture at 20°C for 20 hours, and incubate the culture solution at 4 Centrifuge at 8,000 rpm for 10 min, discard the supernatant, and collect the precipitate, which is the recombinant E. coli BL21 / pET28a-EH wet cell containing the intracellular expression recombinant plasmid. Freeze-dried cells were obtained after the wet cells were freeze-dried for 4 hours. The formula of 1000ml LB medium is: peptone 10g, sodium chloride 10g, yeast extract 5g. After breaking the wet bacteria, centrifuge and di...
Embodiment 3
[0032] Embodiment 3: the application of this epoxy hydrolase in the preparation R-phenyl glycidyl ether
[0033] The freeze-dried thallus obtained in Example 2 was used as a catalyst. Dissolve 50 mg of lyophilized bacteria in 45 mL of 100 mM Trish-HCl, mix thoroughly at 200 rpm for 5 min, then add 5 ml of racemic phenyl glycidyl ether dissolved in 4M DMSO, and stop the reaction at 200 rpm at 30°C for 1 h. Use 15ml of n-hexane to extract three times to get ee>99% R-phenylglycidyl ether, then extract with 15ml ethyl acetate three times to get ee>83%S-phenoxyethylene glycol.
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