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Recombinant halohydrin dehalogenase, encoding gene, vector, engineering bacteria, and applications of recombinant halohydrin dehalogenase

A technology of halohydrin dehalogenase and coding gene, which is applied in the field of recombinant halohydrin dehalogenase, can solve the problems of low catalytic efficiency and poor thermal stability tolerance, and achieve the effect of high catalytic activity and good industrial application prospects

Active Publication Date: 2015-07-15
ZHEJIANG UNIV OF TECH
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  • Claims
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Problems solved by technology

The wild-type HheC has low catalytic efficiency, poor thermal stability and poor tolerance to substrates. Codexis used the protein sequence-activity correlation (ProSAR)-driven protein directed evolution strategy to transform HheC. After 18 years After rounds of mutations, a mutant strain with very high activity was obtained, and its catalytic activity of (S)-CHBE was about 4000 times that of the wild-type enzyme

Method used

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  • Recombinant halohydrin dehalogenase, encoding gene, vector, engineering bacteria, and applications of recombinant halohydrin dehalogenase
  • Recombinant halohydrin dehalogenase, encoding gene, vector, engineering bacteria, and applications of recombinant halohydrin dehalogenase
  • Recombinant halohydrin dehalogenase, encoding gene, vector, engineering bacteria, and applications of recombinant halohydrin dehalogenase

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Experimental program
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Effect test

Embodiment 1

[0021] The acquisition of embodiment 1 parental gene and the preparation of recombinant expression plasmid and recombinant engineered bacteria

[0022] Using the halohydrin dehalogenase (Hhe B) reported in the literature as a probe, the homologous amino acid sequence was searched in the NCBI database, and it was found that the sequence was similar to the short-chain dehydrogenase predicted to be Idiomarina salinarum included in Genbank (Genbank No.KFZ32061. 1) The homology is 45%. Through sequence comparison, it is found that the short-chain dehydrogenase has the same catalytic triplet and some key conserved sequences as the halohydrin dehalogenase. It is speculated that the hydrolyzed protein is a suspected halohydrin dehalogenase enzyme. Because NCBI published the gene sequence of the short-chain dehydrogenase and the codon preference of heterologous expression in Escherichia coli, a synthetic gene was designed and delivered to Shanghai Xuguan Biotechnology Development Co., ...

Embodiment 2

[0024] Embodiment 2 Expression of recombinant halohydrin dehalogenase

[0025] The recombinant Escherichia coli (E.coli) BL21(DE3) / pET28b-HHDH obtained in Example 1 Is , inoculated into LB medium containing a final concentration of 50 mg / L kanamycin sulfate, cultured with shaking at 37°C overnight, and inserted into a 500mL Erlenmeyer flask containing 100mL LB medium at an inoculum size of 1% (v / v) , placed on a shaker at 37°C and 180rpm for shaking culture, when the OD 600 of the culture medium reached 0.6, added IPTG with a final concentration of 0.5mM as an inducer, induced at 28°C for 10h, centrifuged the culture medium, and collected cells (that is, wet cells ), and washed twice with phosphate buffered saline to obtain resting cells. Suspend the obtained resting cells in 20 mL of phosphate buffer solution with pH 8.0, ultrasonically disrupt in an ice bath for 15 min (power 400W, break for 2 seconds and stop for 1 second), and collect the supernatant by centrifugation, wh...

Embodiment 3

[0026] Example 3: Synthesis of (R)-4-cyano-3-hydroxybutyrate ethyl from 50 g / L (S)-4-chloro-3-hydroxybutyrate catalyzed by halohydrin dehalogenase

[0027] With the recombinant Escherichia coli BL21(DE3) / pET28b-HHDH containing the intracellular expression recombinant plasmid obtained in Example 2 Is Wet cells were used as transformation enzymes (E.coli.BL21(DE3), E.coli.BL21(DE3) / pET28b and uninduced E.coli.BL21(DE3) / pET28b-HHDH Is As a control), with (S)-4-chloro-3-hydroxybutyric acid ethyl ester as a substrate, the conversion reaction was carried out to prepare (R)-4-cyano-3-hydroxybutyric acid ethyl ester. The composition of the transformation system and the transformation operation are as follows: add 30mL of 100mM phosphate buffer (pH 7.5) to a 100mL flask, add wet bacteria to make the concentration of the bacteria 10g / L buffer, and use a constant pH / potentiometric titrator to add NaCN to adjust When the pH reached 7.5, (S)-4-chloro-3-hydroxybutyric acid ethyl ester with...

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Abstract

The present invention discloses a recombinant halohydrin dehalogenase, an encoding gene, a recombinant expression vector containing the gene, a recombinant bacterial containing the gene, and applications of the recombinant halohydrin dehalogenase in catalysis synthesis of (R)-4-cyano-ethyl 3-hydroxybutyrate through dehalogenation of (S)-4-chloro-ethyl 3-hydroxybutyrate. Compared with other halohydrin dehalogenases, the halohydrin dehalogenase obtained through the method has characteristics of high catalytic activity, high concentration substrate and product tolerance, and industrial application prospects.

Description

(1) Technical field [0001] The invention relates to a recombinant halohydrin dehalogenase, a coding gene, a carrier, a recombinant genetically engineered bacterium, and the key chiral intermediate (R)-4-cyano-3 for preparing atorvastatin by the recombinant halohydrin dehalogenase - Ethyl hydroxybutyrate application. (2) Background technology [0002] Halohydrin dehalogenases, also known as halohydrin-hydrohalide lyases, catalyze the conversion of aromatic or aliphatic ortho-halohydrins to epoxides and hydrogen halides through an intramolecular nucleophilic substitution mechanism. Halohydrin dehalogenase can not only catalyze the cleavage of carbon-halogen bonds for dehalogenation reactions, but also catalyze and accept a series of unnatural nucleophiles other than halide ions with high selectivity, such as N 3 - , NO 2 - 、CN - etc. mediated epoxide ring-opening reactions. Halohydrin dehalogenase mainly forms a hydrogen bond between the conserved serine in the protein s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/00
CPCC12N9/88C12P7/62C12P13/002C12Y405/01
Inventor 柳志强郑裕国薛锋朱杭芹王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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