Aldo-keto reductase and application thereof

A reductase, aldehyde and ketone technology, applied in the direction of oxidoreductase, the use of carriers to introduce foreign genetic material, viruses/bacteriophages, etc., can solve the problems of low product concentration, low enzyme catalytic activity, poor substrate tolerance, etc., and achieve high The effect of catalytic performance

Inactive Publication Date: 2019-10-29
天津迪嘉医药技术开发有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the asymmetric synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate was performed using genetically engineered strains, and the highest catalytic concentration of the substrate is only 1 M
[0006] Therefore, there is an urgent need in this field for a novel aldehyde and ketone reductase that can be expressed efficiently and has high catalytic performance to solve the shortcomings of low enzyme catalytic activity, poor substrate tolerance, and low product concentration, thereby comprehensively improving the biological method of asymmetric synthesis (R )-2-Hydroxy-4-phenylbutyrate levels

Method used

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  • Aldo-keto reductase and application thereof
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  • Aldo-keto reductase and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] The establishment of embodiment 1 genetically engineered bacteria

[0035] Using the protein PDB database and NCBI database to screen the aldehyde and ketone reductase gene sequence, obtained the Kluyveromyces marxianus Aldoketone reductase sequence of DMKU3-1042 (Genbank: XP 022677552.1). According to the amino acid sequence of the aldehyde and ketone reductase, combined with the codon preference of Escherichia coli for codon optimization, the gene fragment SEQ ID NO.2 was artificially synthesized and the gene was integrated into the pET21a(+) vector (enzyme Cutting sites are NdeI and XhoI). The glucose dehydrogenase BsGDH (preserved in our laboratory) and the pET21a(+)-KmAKR backbone in the pET28a(+) recombinant plasmid were amplified by PCR technology, and the above two The fragments were integrated to construct the pET21a(+)-KmAKR-BsGDH recombinant plasmid, and the integrated recombinant plasmid was transferred into Escherichia coli BL21(DE3) to establish the al...

Embodiment 2

[0039] Embodiment 2 The cultivation of recombinant escherichia coli

[0040] Inoculate the recombinant Escherichia coli obtained in Example 1 into 20 mL of LB medium containing ampicillin (100 μg / mL) (peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.0), incubate at 37°C for 10 h in a shaker at 200 rpm. Transfer 1% bacterial culture solution to a shake flask filled with 100 mL LB medium, and shake culture under the same conditions. When the cell OD 600 When it reaches 0.5-0.8, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM, and induce the expression in the culture medium at 25°C for 10 h. After culturing, the cells were collected by centrifugation (8000 rpm, 10 min, 4°C), washed twice with phosphate buffer (pH 6.8), and centrifuged again to collect the cells (5000 rpm, 10 min, 4°C) for use. Take a small amount of cells for SDS-PAGE to observe the expression of aldehyde ketone reductase and glucose dehydrogenase (see a...

Embodiment 3

[0041] Example 3 Effects of Different Bacterial Concentrations on the Synthesis of (R)-2-Hydroxy-4-Phenylbutyrate Ethyl Ester

[0042] In a 100 mL reaction vial, add 40 mL of phosphate buffer (pH6.8) with a final concentration of 0.1 M, followed by NADPNa 2 (final concentration of 0.2 mM), glucose (final concentration of 1.2 M) and ethyl 2-oxo-4-phenylbutyrate (final concentration of 0.8 M), recombinant Escherichia coli (final concentration of 80 g / L , 120 g / L, 160 g / L). The reaction temperature was maintained at 30-37°C with a water bath, and the pH was maintained at 6.5-6.8 with a 2 M sodium carbonate solution. The reaction was terminated after 8 h. An equal volume of ethyl acetate was added for extraction, and the organic phase was collected after centrifugation (8000 rpm, 10 min, 4°C). The organic phase was rotary evaporated to a constant volume, and a certain amount of anhydrous sodium sulfate was added to dry overnight to obtain the final product (R)-ethyl 2-hydroxy-4...

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Abstract

The invention relates to analdo-keto reductase, and application of recombinant expression transformant of the aldo-keto reductaseas a biocatalyst in asymmetric reduction of 2-carbonyl-4-phenylbutanoate to synthesis (R)-2-hydroxyl-4-phenylbutyrate, and belongs to the technical field of bioengineering. The amino acid sequence of the aldo-keto reductase is shown in SEQ ID NO.1. The encodinggene the aldo-keto reductase corresponds to the nucleotide sequence of the amino acid sequence of the SEQ ID NO.1 and is shown in SEQ ID NO.2. The novel aldo-keto reductaseis high in catalytic performance.

Description

technical field [0001] The present invention relates to an aldehyde and ketone reductase and its recombinant expression transformant as a biocatalyst to synthesize (R)-2-hydroxy-4-phenylbutyric acid through the asymmetric reduction of 2-carbonyl-4-phenylbutyric acid ethyl ester The application in ethyl ester belongs to the technical field of bioengineering. Background technique [0002] (R)-2-Hydroxy-4-phenylbutyric acid ethyl ester (molecular formula is C 12 h 16 o 3 , with a molecular weight of 208.25 and a CAS number of 90315-82-5) is an important chiral building block for the synthesis of various angiotensin-converting enzyme inhibitor (ACEI) drugs (such as benazepril, cilazapril, etc.). ACEI drugs are mainly used to treat diseases such as hypertension and congestive heart failure. In the antihypertensive drug market, it shares the top three positions with non-peptide angiotensin II receptor inhibitors and calcium channel antagonists. Therefore, research on the chir...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/70C12N1/21C12P7/62C12R1/19
CPCC12N9/0006C12N15/70C12P7/62C12N2800/22
Inventor 王兴初潘硕杜永静秦丽丽夏俊刚
Owner 天津迪嘉医药技术开发有限公司
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