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Stereoselectivity-improved phaseolus-vulgaris epoxy hydrolytic enzyme mutant

A technology of hydrolyzing enzymes and mutants, which is applied in the field of enzyme engineering, can solve the problems that cannot satisfy the production of high-enantiopure epoxides and vicinal diols, and achieve the effect of improving enantioselectivity

Active Publication Date: 2018-09-21
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The present invention adopts the method of whole plasmid PCR to carry out unidirectional site-directed mutation on the 184-position proline, obtains the mutant enzyme P184L with simultaneously improved stereoselectivity and catalytic activity, and solves the problem that the stereoselectivity cannot satisfy the production of high-enantiopure epoxides The demand for vicinal diols lays the foundation for broadening the industrial application of PvEH1

Method used

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  • Stereoselectivity-improved phaseolus-vulgaris epoxy hydrolytic enzyme mutant
  • Stereoselectivity-improved phaseolus-vulgaris epoxy hydrolytic enzyme mutant
  • Stereoselectivity-improved phaseolus-vulgaris epoxy hydrolytic enzyme mutant

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Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Mutant plasmid construction

[0032]The E.coli BL21(DE3) / pET-28a-pveh1 (Ye Huihua, Hu Die, Li Chuang, etc.) preserved from the laboratory using the PurePlasmidMini Kit (purchased from Kangwei Reagent Co., Ltd.). Heterologous expression and enantio-normalized catalytic properties of oxide hydrolase[J]. China Biotechnology, 2016, 36(10): 21-27. 169: 41-54. template,

[0033] The primers used were P184L-F (5'-3'GAGACCAGGACCACTAATACTCCCCAA); P184L-R (5'-3'TTGGGGAGTATTAGTGGTCC TGGTCTC).

[0034] use HS PCR enzyme (purchased from TaKaRa Company) adopts the method of whole plasmid PCR. Using the PvEH1 plasmid as a template, PCR conditions: denaturation at 98°C for 10 s; annealing at 55°C for 5 s; extension at 72°C for 3.5 min, 30 cycles, and extension at 72°C for 10 min. The PCR product was transformed into E.coli BL21 (DE3) competent cells by digesting the template plasmid with Dpn I enzyme, and the transformation was performed according to the instruction man...

Embodiment 2

[0035] Embodiment 2: the acquisition of mutant enzyme

[0036] The obtained mutant enzyme expression vector was inoculated (the inoculum size was 1%) in 2 mL of LB medium containing 1% kanamycin, at 37 ° C, 220 r min -1 Cultivate overnight; take 2mL culture medium and transfer to 100mL LB medium containing 1% kanamycin, cultivate to OD 600 When it is 0.6~0.8, add 100μL of IPTG (500mmol·L -1 ) to a final concentration of 0.5mmol L -1 After induction at 20°C for 10 h, the recombinant bacterial cells were collected by centrifugation. It was determined that the enzyme activity of the epoxide hydrolase of the bacteria to rac-pCSO was 10.0 U / g of the bacteria cells.

Embodiment 3

[0037] Example 3: Enantioselectivity comparison of enzymes before and after mutation to rac-pCSO

[0038] The invention compares the stereoselectivity of the enzyme before and after the mutation, and the stereoselectivity includes two properties of enantioselectivity and enantionormalization. Wherein the wild enzyme refers to PvEH1 derived from Phaseolus vulgaris, GenBank Accession No. XM007146940.

[0039] Use 1ml of sodium phosphate buffer solution (pH=7.0, 100mmol·L) for every 20mg of wet bacteria -1 ) suspension, take 1mL and add it to a 2mLEP tube, add 50μL of 200mmol·L -1 rac-pCSO (solvent is methanol), the final substrate concentration is 10mmol L -1 . Placed at 25°C, 220r·min -1 Shaking table reaction, 50 μL samples were extracted three times with ethyl acetate (1 mL in total) at 30 min, 1 h, 2 h and 24 h respectively, dried with anhydrous magnesium sulfate and analyzed by high performance liquid chromatography. When the conversion rate c was about 50%, the E valu...

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Abstract

The invention discloses a stereoselectivity-improved phaseolus-vulgaris epoxy hydrolytic enzyme mutant, and belongs to the technical field of enzyme engineering. Phaseolus-vulgaris-source epoxy hydrolytic enzyme is subjected to one-way site-specific mutagenesis, and enantioselectivity-and-enantiomer-polarity-improved mutate enzyme P184L is obtained. The final eep of the P184L for racemic m-chlorostyrene oxide (rac-mCSO) is increased to 27.3% from 3.1% at the temperature of 25 DEG C. Compared with a wild type, the E value of the P184L for racemic p-chloro styrene oxide (rac-pCSO) is increasedto 20 from 2.6, and the enantioselectivity of the rac-pCSO is increased by 7.7 times.

Description

technical field [0001] The invention relates to a mutant of kidney bean epoxyhydrolase with improved stereoselectivity and a construction method, belonging to the technical field of enzyme engineering. Background technique [0002] Enantiopure p-chlorostyrene oxide (pCSO) and its hydrolysis product p-chlorophenylethylene glycol (pCPED) are important intermediates for various chiral drugs and functional materials, such as R-type p-chlorostyrene oxide ( (R)-pCSO) can be used for the synthesis of CB1 antagonists; R-type p-chlorophenylethylene glycol ((R)-pCPED) is an important chiral building block of NMDA receptor inhibitor - ilirodine. Currently, a series of biochemical methods for the synthesis of enantiopure pCSO and pCPED have been reported. Among them, Suresh et al. used a class of chiral macrocyclic Schiff bases to catalyze the oxygenation of p-chlorostyrene to synthesize (R)-pCSO, and its ee p Up to 47%; Karboune et al. used epoxide hydrolase (AnEH) from recombinant A...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N15/55
CPCC12N9/14C12Y303/02
Inventor 邬敏辰宗迅成徐雄峰王瑞李雪晴李剑芳
Owner JIANGNAN UNIV
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