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Epoxide hydrolase from rhodosporidium paludigenum and application of epoxide hydrolase

A technology of marine red yeast and epoxide, applied in the field of bioengineering, can solve the problem that the theoretical yield is only 50%, and achieve large-scale industrial production and application potential and economic value, high enantioselectivity and catalytic activity Effect

Active Publication Date: 2019-02-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, about 40 EHs with high enantioselectivity (enantioselectivity E value > 30) have been reported, which are mainly used in the hydrolytic kinetic resolution of epoxides, but the theoretical yield is only 50%.

Method used

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  • Epoxide hydrolase from rhodosporidium paludigenum and application of epoxide hydrolase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Cloning of RPEH1 mature peptide cDNA sequence

[0023] (1) A pair of specific primers were designed according to the highly homologous marine red yeast (Rhodosporidium paludigenum) CBS 6565 epoxide hydrolase gene sequence, the primer sequence is as follows:

[0024] RpEH1-F: 5'-CATATGGCTGCCCATTCCTTTACTGC-3', containing Nde I restriction site

[0025] RpEH1-R: 5'-GAATTCTCAGGCCTGGTTCGACAGCAA-3', containing EcoR I restriction site

[0026] The total RNA of R.paludigenum obtained by screening was extracted, and RT-PCR was performed according to the instructions of TaKaRa RNA PCR Kit (Ver.3.0). The first strand of cDNA was synthesized by reverse transcription using Oligo dT-Adaptor Primer as a primer. The first round of PCR amplification was performed with RpEH1-F and M13Primer M4 as primers. The PCR conditions were: 94°C for 5 min; 94°C for 30 s, 51°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 10 min. The second round of PCR amplification was carried out usi...

Embodiment 2

[0027] Example 2 Expression of RPEH1 mature peptide gene in E.coli BL21(DE3)

[0028] The pUCm-T-Rpeh1 and pET-28a(+) plasmids with correct sequencing results were double-digested with Nde I and Not I, and the digested products recovered from rubber tapping were ligated under the action of T4 DNA ligase to obtain the recombinant plasmid pET -28a(+)-Rpeh1, and sequenced the recombinant plasmid. Transform pET-28a(+)-Rpeh1 into E.coli BL21(DE3), and after screening on the resistance plate containing Kan, pick the positive transformants and put them in 2mL of Kan-containing resistant LB liquid medium at 37°C 215r / min shaker shaking culture 14 ~ 16h. Transplanted in 30mL of the same medium at 1% inoculum size, cultured on a shaker at 215r / min at 37°C until mid-logarithmic growth (OD 600 =0.6-0.8), adding IPTG with a final concentration of 150 μM, and inducing culture at 16° C. at 215 r / min for 10 h. The fermentation broth was centrifuged to collect the bacterial cells, washed tw...

Embodiment 3

[0029] Example 3 Application of RPEH1

[0030] The whole cell freeze-dried enzyme powder obtained by the method in Example 2 was resuspended in 50 mM pH7.2 phosphate buffer, so that the cell concentration of the resuspended bacteria was 100 mg / mL. Add 100 μL of the aforementioned resuspended bacteria solution, 45.6 μL of styrene oxide stock solution, and 854.4 μL of 50 mM phosphate buffer saline at pH 7.2 to a 2 mL EP tube. React at 25°C and 220rpm, take samples regularly for gas chromatographic analysis to calculate the conversion rate and ee p value. The result shows that under the final concentration of 10mg / mL bacterial concentration, reacted for 6 hours, the substrate styrene oxide was completely hydrolyzed, and the conversion rate reached 100%, ee p to 67%.

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Abstract

The invention discloses an epoxide hydrolase from rhodosporidium paludigenum and application of the epoxide hydrolase, and belongs to the technical field of biology. The epoxide hydrolase and the application have the advantages that epoxide hydrolase genes (rpeh1) from the rhodosporidium paludigenum R. paludigenum and corresponding amino acid sequences are provided; recombinant plasmids pET28a(+)-rpe1 with the genes and genetically engineered bacteria E. coli BL21 (DE3) / pET28a(+)-rpeh1 are constructed, and recombinant epoxide hydrolases are prepared by the aid of bacterial strains; biologicalcatalysis can be carried out by the constructed genetically engineered bacteria, and accordingly the ee of enantiomerically pure R-type phenyl glycol obtained by means of preparation is higher than 73%.

Description

technical field [0001] The invention relates to an epoxide hydrolase derived from marine rhodotorula and application thereof, belonging to the technical field of bioengineering. Background technique [0002] Chiral epoxides or vicinal diols are a class of high value-added multifunctional synthons that can be used in the synthesis of drugs, fine chemicals, pesticides, and functional materials. Epoxide hydrolases (EHs) can catalyze the hydrolytic kinetic resolution or enantionormalized hydrolysis of racemic epoxides, retaining single-configuration epoxides or converting them into chiral vicinal diols. About 40 EHs with high enantioselectivity (enantioselectivity E value > 30) have been reported, which are mainly used in the hydrolytic kinetic resolution of epoxides, but the theoretical yield is only 50%. [0003] Unlike hydrolytic resolutions, the theoretical yields of enantionormalized hydrolysis can be as high as 100%. There are three ways to prepare chiral vicinal diol...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55C12P7/22
CPCC12N9/14C12P7/22C12Y303/02003
Inventor 徐雄峰王婷婷苏永君章程胡博淳胡蝶李剑芳邬敏辰
Owner JIANGNAN UNIV
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