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Recombinant ketoreductase and application thereof to preparation of (R)-3-hydroxybutyric acid and salt thereof

An R-3-, hydroxybutyric acid technology, applied in the field of recombinant protein, can solve the problems of difficult separation and purification, difficult industrial application, low fermentation unit, etc., and achieve breakthroughs in low chiral purity, low production cost, and high cost reduction. Effect

Active Publication Date: 2019-06-07
洛阳华荣生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Shenzhen Ecoman Biotechnology uses genetic engineering Escherichia coli fermentation method to prepare R-3HB (CN200910106660.7), but its fermentation unit is low, cost is high, separation and purification are difficult, and it is difficult for industrial application
Although there are no research reports in this area, there are many studies on the reduction of ethyl 4-chloroacetoacetate to obtain ethyl 4-chloro-3-hydroxybutyrate

Method used

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  • Recombinant ketoreductase and application thereof to preparation of (R)-3-hydroxybutyric acid and salt thereof
  • Recombinant ketoreductase and application thereof to preparation of (R)-3-hydroxybutyric acid and salt thereof
  • Recombinant ketoreductase and application thereof to preparation of (R)-3-hydroxybutyric acid and salt thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] (1) Wild CmCR Ketoreductase Engineering Bacteria

[0072] The ketoreductase shown in SEQ ID NO: 1 (GenBank: AB036927, derived from the nucleotide sequence of Candida magnoliae) was synthesized through the whole gene sequence, and then connected with the plasmid pET28a (commercially available) to obtain the recombinant The vector pET28a-CmCR; then the recombinant vector was introduced into Escherichia coli BL21(DE3) to construct the Escherichia coli genetic engineering strain pET28a-CmCR / BL21(DE3) expressing ketoreductase.

[0073] The amino acid sequence of wild ketoreductase is shown below:

[0074] MAKNFSNVEYPAPPPAHTKNESLQVLDLFKLNGKVASITGSSSGIGYALAEAFAQVGADVAIWYNSHDATGKAEALAKKYGVKVKAYKANVSSSDAVKQTIEQQIKDFGHLDIVVANAGIPWTKGAYIDQDDDKHFDQVVDVDLKGVGYVAKHAGRHFRERFEKEGKKGALVFTASMSGHIVNVPQFQATYNAAKAGVRHFAKSLAVEFAPFARVNSVSPGYINTEISDFVPQETQNKWWSLVPLGRGGETAELVGAYLFLASDAGSYATGTDIIVDGGYTLP

[0075] The nucleotide sequence of wild ketoreductase is as follows:

[0076] ATGGCTAAGAA...

Embodiment 2

[0084] Example 2 Construction of Ketoreductase Saturation Mutant Library

[0085] (1) Computer simulation of CmCR ketoreductase structure modeling

[0086] CmCR derived from Candida magnoliae is homologous to carbonyl reductase (PDB: 3CTM) (CACR for short) derived from Candida parapsilosis. Homologous modeling was carried out by Sybyl software to obtain its 3D structure ( figure 2 a). According to the 3D structure diagram of homology modeling, it was found that the three sites S176, Y191, and K195 in CmCR played an important role in the reaction with ethyl acetoacetate as the substrate ( figure 2 b).

[0087] (2) Molecular Construction of Saturation Mutation Library

[0088] According to the computer three-dimensional structure simulation results, S176, Y191, and K195 were subjected to saturation combination mutations to establish a saturation mutation gene bank. The specific method is as follows:

[0089] ①Primer design

[0090] Using the sequence of SEQ ID NO: 1 as a ...

Embodiment 3

[0125] The fermenter fermentation of embodiment 3 recombinant strains

[0126] (1) Recombinant ketoreductase mutant strain CmCR-186 / BL21 (DE3) 7 liter fermenter fermentation

[0127] Inoculate the single clone of the recombinant ketoreductase mutant strain CmCR-186 / BL21(DE3) into 4 mL of LB medium containing 50 mg / L kanamycin resistance, culture at 37°C and 200 rpm for 20 h, and then take 2 mL of the overnight culture The cells were transferred to 200mL seed medium, and after culturing at 37°C and 200rpm for 4h, all the 200mL cultured cells were inoculated into a 7L fermenter containing 4.8L fermentation medium for fermentation. Fermentation tank culture conditions: tank temperature 37°C, aeration ratio 2.5vvm, stirring 350rpm, tank pressure 0.05MPa, when the OD600 of the bacteria in the fermentation broth = 5-6, add ITPG with a final concentration of 0.2mM for induction, and lower the temperature to 25°C , the tank pressure, air flow, etc. remain unchanged, continue to culti...

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Abstract

The invention relates to recombinant protein, in particularly to recombinant ketoreductase and an application thereof to preparation of (R)-3-hydroxybutyric acid and salt thereof. The recombinant ketoreductase contains (1) an amino acid sequence as shown in SEQID NO:1, wherein one or more equivalent amino acid residues in the 176th amino acid, the 191st amino acid and the 195th amino acid are substituted; or (2) an amino acid sequence as shown in (1), wherein one or more amino acids except those at the 176th site, the 191st site and the 195th site are substituted, deleted, added and / or inserted. Compared with wild type ketoreductase, the recombinant ketoreductase has the advantage that the enzyme activity is increased. The activity of the recombinant ketoreductase on ethyl acetoacetate substrates is improved by at least 300% than that of the wild type ketoreductase, wherein the activity of a CmCR-186 mutant on the ethyl acetoacetate substrates is improved by 879% than that of the wildtype ketoreductase.

Description

technical field [0001] The invention relates to a recombinant protein, in particular to a recombinant ketoreductase and its application in the preparation of R-3-hydroxybutyric acid and its salt. Background technique [0002] R-3-hydroxybutyric acid (R-3HB, figure 1 ), also known as β-hydroxybutyric acid, its English name is (R)-3-hydroxybutyric acid or (R)-3-hydroxybutyrate. R-3-hydroxybutyric acid is a hygroscopic monoclinic crystal with a melting point of 49-50°C and a specific optical rotation of -24.9°. It is soluble in water, ethanol, ether, and insoluble in benzene. Since 3HB has publicized hydroxyl and carboxyl groups, it can be Perform various typical reactions of the two groups. [0003] 3-Hydroxybutyric acid is not only an important monomer for the synthesis of PHA, but also has important significance in the living body. It is one of the important components of ketone bodies in mammals, accounting for about 10% of fatty acids in the liver. 70% of ketone bodies ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/70C12N1/21C12P7/42
Inventor 范文超丁鹏陈涛王庆培
Owner 洛阳华荣生物技术有限公司
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