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Halohydrin dehalogenase mutant and application thereof

A halohydrin dehalogenase and mutant technology, applied in the biological field, can solve the problems of excellent catalytic performance, inability to meet industrial production, and few industrial production.

Pending Publication Date: 2020-01-17
YANCHENG INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since 2014, although the number of halohydrin dehalogenases has been increasing, there are fewer enzymes with excellent catalytic performance. In recent years, only HheC from Agrobacterium radiobacter AD1 has reported excellent catalytic performance.
[0006] Although halohydrin dehalogenases are widely used, most natural enzymes have great limitations, such as low enantioselectivity, and cannot meet the needs of industrial production, so that they are rarely directly applied in industrial production

Method used

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  • Halohydrin dehalogenase mutant and application thereof
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  • Halohydrin dehalogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0047] Example 2. Homology Modeling

[0048] Using the crystal structures of HheC from Agrobaterium radiobater AD1 and HheB from Corynebacteriumsp.N-1074 as templates, Modeller 9.18 was used for HHDH His The three-dimensional structure was homologously modeled, and then CHARMM27 was used to optimize the model. The evaluation program ProCheck was used to evaluate the model to evaluate the reliability of the obtained model. The modeling results were analyzed using the software PyMOL 1.2rl. Ended up with high quality HHDH His structural model. HHDH His The evaluation results of the model showed that: 89.8% were located in the optimal region, 8.2% were located in other allowed regions, 2.0% were located in the general allowed region, and no residues were located in the disallowed region, which indicated that the model was reliable. Through sequence alignment and structural analysis, it was found that HHDH His The catalytic triad structure is Ser116-Tyr129-Arg133.

Embodiment 3

[0049] Example 3. Molecular docking

[0050] Using the molecular docking software Autodock 4.0, the halohydrin dehalogenase HHDH His Molecular docking to the active center of racemic PGE; select distance from substrate molecule Nearby sites were subjected to site-directed saturation mutagenesis, and the final selected mutation sites were Gln159, Asn160, Phe161, Tyr167, Phe168, and Pro169. The primers used for site-directed mutagenesis are shown in Table 3.

[0051] Table 3 PCR primers used for mutation

[0052]

[0053] N stands for A, T, G or C

[0054] Site-directed saturation mutation was performed on the selected 6 sites, and clones were selected for activity screening at each site. First, false positive strains with no activity were eliminated through high-throughput screening, and then mutant strains with improved stereoselectivity were screened through liquid phase detection. by HHDH His Screening of the 10 saturation mutant library, at the Phe161, Tyr167, and P...

Embodiment 4

[0055] Example 4. Induced expression of halohydrin dehalogenase and mutants thereof

[0056] HHDH His and its mutants N160L and Q159L were induced to express. Cultivate the recombinant halohydrin dehalogenase engineered bacteria at 37°C and 200r / min for 10h, add the Escherichia coli with the recombinant expression plasmid to 50mL LB liquid medium at an inoculum size of 1%, and add a final concentration of 50mg / min. L of kanamycin, cultured to OD at 37°C and 200r / min 600 If the value is 0.6-0.8, IPTG inducer is added to make the final concentration 0.15mM to induce its protein expression. Induce the culture at 28°C and 200r / min for about 10h, centrifuge at 5000×g for 5min, collect the cells, resuspend the bacteria with pH 8.0 phosphate buffer (200mM), and ultrasonically disrupt the cells.

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Abstract

The invention first discloses a halohydrin dehalogenase mutant and application thereof. According to the invention, a database mining technology is used to mine potential halohydrin dehalogenase genes, and the halohydrin dehalogenase genes are induced to express in engineering bacteria to obtain a new halohydrin dehalogenase. Semi-rational design and modification of the enzyme is performed, and (R, S)-PGE is used as a model substrate to obtain two mutants with improved stereoselectivity, which are Q159L and N160Lrespectively. The enantioselectivity of the two mutants is increased approximatelyfrom 9.85 to 21.80 and 21.10respectively. The selectivity of the mutant N160L is opposite to selectivity of the original enzyme. The selectivity to various substrates of the two mutants is improved to a certain extent. In particular, the E value of the mutant N160L for the substrate P-methylphenyl glycidyl ether is increased to 25.77, which is 13.93 times greater than E value of the original HHDHHis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a halohydrin dehalogenase mutant and application thereof. Background technique [0002] As important intermediates in pharmaceutical and chemical industries, halohydrin compounds are widely used in organic synthesis and pharmaceutical research and development. However, most halides in nature have disadvantages such as poor degradation ability, high toxicity and potential carcinogenicity. It has become one of the main environmental pollutants, so people pay more and more attention to its degradation treatment in order to reduce the pollution of this kind of organic halides to the natural environment and the potential threat to human beings. The use of traditional physical and chemical methods not only has harsh reaction conditions, but also may cause secondary pollution, while the use of biodegradation methods has outstanding advantages such as no pollution to the environm...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P41/00C12P17/02C12P7/22C12R1/19
CPCC12N9/88C12Y405/01C12N15/70C12P41/002C12P17/02C12P7/22
Inventor 薛锋李凤伟亚香菊王于齐
Owner YANCHENG INST OF TECH
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