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Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof

A technology that encodes genes and reductases, applied in genetic engineering, oxidoreductases, applications, etc., can solve problems such as reducing catalytic efficiency, and achieve the effects of efficient circulation, high optical purity, and specific stereoselectivity

Inactive Publication Date: 2016-06-15
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The industrial application of asymmetric synthesis of (S)-N-Boc-3-piperidinol requires the application of high-concentration substrates, and high-concentration substrates and by-substrates and high-concentration products will inhibit enzyme activity. thereby reducing the catalytic efficiency

Method used

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  • Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof
  • Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof
  • Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction and induced expression of recombinant carbonyl reductase ReCR Escherichia coli genetically engineered bacteria

[0034] (1) Recombinant carbonyl reductase ReCR Escherichia coli genetic engineering bacteria

[0035] Using the genomic DNA of Rhodococcus erythropolis WZ010 strain as a template, the designed primers F1 and R1 were used for PCR amplification. The amplification system is shown in Table 1.

[0036] Table 1 PCR amplification reaction system

[0037]

[0038] The primers are as follows: F1, 5'-ATGAAGGCAATCCAGTACAC-3'; R1, 5'-CTACAGACCAGGGACCACA-3'. The PCR reaction process is as follows: pre-denaturation at 94°C for 5 minutes; after that, denaturation at 94°C for 30 s, renaturation at 55°C for 30 s, and 72°C for 1 min as a cycle, repeating this cycle 30 times; finally, 72°C for 10 min. PCR products were detected by 0.8% agarose gel electrophoresis. figure 2 A bright band can be seen at about 1000bp, which is consistent with the the...

Embodiment 2

[0045] Embodiment 2: the activity measurement of recombinant carbonyl reductase ReCR and its genetically engineered bacteria

[0046] Enzyme activity was determined by spectrophotometry, and the change in absorbance at 340nm was measured at the optimum temperature and pH, and repeated twice; the optimum temperature and pH for alcohol oxidation were 50°C and 10.0, respectively, while the optimum for ketone reduction was 50°C and 10.0, respectively. The temperature and pH were 60°C and 6.0, respectively. Standard reducing activity assay system (2.5 mL): 50 mMPIPES buffer (pH 6.0), 10 mM N-Boc-3-piperidone, 0.4 mM NADH, recombinant carbonyl reductase ReCR enzyme solution prepared by the method in Example 1 2 μg / mL. Standard oxidation activity assay system (2.5mL): 50mM CAPSO buffer (pH10.0), 50mM 2-octanol, 0.4mM NAD + , the recombinant carbonyl reductase ReCR enzyme solution prepared by the method in Example 1 was 2 μg / mL. Enzyme activity unit definition: at the optimum reacti...

Embodiment 3

[0048] Embodiment 3: Optimum, pH optimum temperature and thermostability of recombinant carbonyl reductase ReCR

[0049] (1) adopting the common buffer system configuration buffer solution of 50mM in the concentration of different pH buffer ranges: MES buffer solution, pH=5.5,6.0; PIPES buffer solution, pH=6.1,6.5,7.0,7.5; Tris-HCl buffer solution, pH=7.5, 8.0, 8.5, 9.0; CAPSO buffer, pH=9.0, 9.5, 10.0; CAPS buffer, pH=10.0, 10.5, 11.

[0050] Reducing activity assay system (2.5 mL): 50 mM buffer, 10 mM N-Boc-3-piperidone, 0.4 mM NADH, recombinant carbonyl reductase ReCR enzyme solution prepared by the method in Example 1 2 μg / mL. Oxidation activity assay system (2.5mL): 50mM buffer, 50mM 2-octanol, 0.4mM NAD + , the recombinant carbonyl reductase ReCR enzyme solution prepared by the method in Example 1 was 2 μg / mL. The reaction temperature for alcohol oxidation is 50°C, while the reaction temperature for ketone reduction is 60°C. The activities of the recombinant carbonyl ...

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Abstract

The invention discloses a recombinant carbonyl reductase ReCR, a coding gene, a carrier, an engineering bacterium and its application in the biological enzymatic preparation of (S)-N-Boc-3-piperidinol. The recombinant carbonyl reductase ReCR The amino acid sequence of the amino acid sequence is shown in SEQ ID NO.2; the method of the present invention uses the recombinant carbonyl reductase ReCR of high activity, stereoselective specificity, and irreversible reduction of N-Boc-3-piperidone as a biocatalyst, so that The reaction balance is beneficial to the formation of (S)-N-Boc-3-piperidinol, and the product has high optical purity; using 2-octanol as the cosubstrate, the efficient cycle of coenzyme is realized; using 2-octanol as the organic phase to construct two The phase catalysis system, combined with the whole cell catalysis method, greatly reduces the inhibition of enzyme activity by high-concentration substrates, by-substrates and products. The applicable substrate concentration is as high as 200g / L, and the reaction time is <12h. The product (S)-N The yield of -Boc-3-piperidinol was 94.17%, and the e.e. value of the product was >99%.

Description

(1) Technical field [0001] The present invention relates to a preparation method of (S)-N-Boc-3-piperidinol, in particular to a method for the preparation of chiral pure N-Boc-3-piperidinone by biological enzymatic asymmetric reduction (S)-N-Boc-3-piperidinol method. (2) Background technology [0002] Many drug molecules contain nitrogen heterocyclic structural units. Analysis of drug data approved by the US FDA shows that 59% of small molecule drugs contain nitrogen heterocycles, the most common of which is a six-membered ring; among the top 25 most commonly used nitrogen heterocycle drugs, piperazine ranks first Drugs with pyridine structure. (S)-N-Boc-3-piperidinol is an important chiral nitrogen-containing saturated heterocyclic alcohol with active functional groups "-OH" and "-NH-" Important chiral dicing. Natural medicines with high anti-malarial activity, hemosine and anomaline, the antagonist of neurokinin receptors L-733,060, and the inhibitor of cyclin-dependen...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C12N1/21C12P17/12C12R1/19
CPCC12N9/0006C12P17/12C12Y101/01184
Inventor 应向贤汪钊黄美娟范雅君
Owner ZHEJIANG UNIV OF TECH
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