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Marine bacterial novel esterase, as well as preparation method and application thereof

An esterase, a consistent technology, applied in the field of marine bacterial new esterase and its preparation and application, can solve the problems of expensive commercial enzymes and unsuitable for large-scale industrial production, etc.

Active Publication Date: 2013-07-24
SECOND INST OF OCEANOGRAPHY MNR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Recently, Liu et al. (PRocess Biochem.47(2012), 1037-1041.) used immobilized lipase (Novozym435) from Candida antaRctica to catalyze the reaction to obtain (R)-3-(4-fluorophenyl)pentane Diacid monomethyl ester, although the yield and ee value are high, but due to the high price of commercial enzymes, it is not suitable for large-scale industrial production

Method used

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  • Marine bacterial novel esterase, as well as preparation method and application thereof
  • Marine bacterial novel esterase, as well as preparation method and application thereof
  • Marine bacterial novel esterase, as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: the construction of the recombinant expression plasmid of esterase gene pe8 and recombinant strain

[0046] Pelagibacterium halotolerans B2 T The middle esterase gene pe8 was cloned into the expression vector, and the recombinant expression strain was constructed. Based on the core conserved sequences of all esterase families published so far, the upstream primer pe8F (5'-AGGA CATATG ACCGAACCCGTAAAG-3', Nde I) and downstream primer pe8R (5'-CGAT AAGCTT CTAGAGGATCTCGCG-3', HindIII), PCR amplification confirmed the full-length sequence of the gene. The expression plasmid was constructed by enzyme digestion cloning, that is, the PCR product was double-digested with NdeI and HindIII, the digested fragment was recovered by tapping the rubber, and it was ligated with the plasmid pET28b(+) that had also been double-digested with NdeI and HindIII. 2 The transformation method was transformed into Escherichia coli DH5α, and positive clones were screened for kana...

Embodiment 2

[0047] Embodiment 2: Utilize recombinant expression strain to express recombinant esterase gene pe8

[0048] Transfer 3ml of the constructed recombinant expression strain to 100ml of LB liquid medium containing 20μg / ml kanamycin and 34μg / ml chloramphenicol, and shake at 37°C until OD 600 When it reaches 0.6, add IPTG with a final concentration of 0.5mM to induce expression, and transfer to 25°C for shaking culture overnight. The cells were collected by low-temperature centrifugation and resuspended in PBS buffer (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , pH7.4), washed twice, and ultrasonicated on ice. The supernatant was collected by low-temperature centrifugation, and freeze-dried for 24 hours to obtain PE8 esterase powder.

Embodiment 3

[0049] Example 3: Hydrolysis of 3-(4-fluorophenyl) dimethyl glutarate by esterase PE8

[0050] Use esterase PE8 to hydrolyze 3-(4-fluorophenyl) dimethyl glutarate, specific operation: Include 40mM 3-(4-fluorophenyl) dimethyl glutarate, 5mg PE8 in a 0.5ml reaction system Enzyme powder, 30°C, 200rpm shaking reaction for 24h, adjust the pH to 2.0 with 5M hydrochloric acid to terminate the reaction, add 0.5ml ethyl acetate to extract twice, vacuum dry to remove ethyl acetate, then add 300μl isopropanol to dissolve the precipitate, high-efficiency solution The product components were analyzed by phase chromatography.

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Abstract

The invention discloses a marine bacterial novel esterase, and a method for producing a drug intermediate (R)-3-(4-fluorophenyl) monomethyl glutarate by chirally catalyzing 3-(4-fluorophenyl) methyl glutarate by using the esterase. The gene of the esterase is cloned to an expression plasmid to transform escherichia coli Rosetta. As the esterase can be highly and solubly expressed in an expression strain, and shows excellent salt resistant, alkali resistance and chiral selectivity, the esterase can be used as potential enzyme for industrial production of the antidepressant drug intermediate (R)-3-(4-fluorophenyl) monomethyl glutarate. As long as the reaction conditions are optimized, the esterase can be used for catalyzing 3-(4-fluorophenyl) methyl glutarate to produce the drug intermediate (R)-3-(4-fluorophenyl) monomethyl glutarate; and as a result, the transformation ratio and chiral selectivity of the esterase are greatly improved.

Description

technical field [0001] The invention relates to a novel esterase of marine bacteria, a preparation method and application thereof. Specifically, the marine bacterium Pelagibacterium halotolerans B2 T Middle esterase gene pe8 and use of the chiral esterase to catalyze 3-(4-fluorophenyl)glutaric acid dimethyl ester to produce pharmaceutical intermediate (R)-3-(4-fluorophenyl)glutaric acid monomethyl ester method. Background technique [0002] Optically pure (R)-3-(4-fluorophenyl)glutaric acid monomethyl ester is the precursor of a series of important pharmaceutical intermediates. One of the most representative is levo-paroxetine hydrochloride. It can increase the concentration of 5-hydroxytryptamine (5-HT) in the synaptic gap, play an antidepressant effect, have weak effects on other transmitters, and have little effect on the autonomic nervous system and cardiovascular system. Serotonin (5-HT) reuptake inhibitors (SSRIs). Clinically, it is widely used to treat diseases s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N1/21C12N1/19C12N5/10C12P7/62
Inventor 吴月红江夏薇许学伟魏笑莲王春生吴敏
Owner SECOND INST OF OCEANOGRAPHY MNR
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