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A method for enzymatic asymmetric reduction of citral to improve the optical purity of (r)-citronellal

A technology of optical purity and citral, applied in fermentation and other fields, can solve the problems of expensive catalyst and low yield

Active Publication Date: 2019-10-29
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The industrial synthesis of L-menthol by this chemical method has been used for many years, but there are still problems such as expensive catalyst and low yield.

Method used

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  • A method for enzymatic asymmetric reduction of citral to improve the optical purity of (r)-citronellal
  • A method for enzymatic asymmetric reduction of citral to improve the optical purity of (r)-citronellal
  • A method for enzymatic asymmetric reduction of citral to improve the optical purity of (r)-citronellal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Expression and purification of Saccharomyces cerevisiae enol reductase OYE1 and formate dehydrogenase FDHCB from Candida boidinii

[0034] (1) Construction and induced expression of Saccharomyces cerevisiae enol reductase OYE1 Escherichia coli genetically engineered bacteria

[0035] Saccharomyces cerevisiae enol reductase OYE1 gene oye1 was amplified by PCR with the designed primers F1 and R1 using Saccharomyces cerevisiae CICC 1002 genomic DNA purchased from China Industrial Microorganism Culture Collection Management Center. Table 1 shows.

[0036] Table 1 PCR amplification reaction system

[0037]

[0038] The primers are as follows: F1, 5'-ATGCCATTTGTTAAGGACTTTA-3'; R1, 5'-TTAATTTTTGTCCCAACCGA-3'. The PCR reaction process is as follows: pre-denaturation at 94°C for 5 minutes; then, a cycle of denaturation at 94°C for 30 s, renaturation at 57°C for 30 s, and holding at 72°C for 1 min, repeating this cycle 35 times; finally, holding at 72°C for 10 min...

Embodiment 2

[0045] Embodiment 2: the enzyme activity assay of Saccharomyces cerevisiae enol reductase OYE1

[0046] The standard enzyme activity assay system (2mL) contains 0.4mM NADH, 100μg / mL Saccharomyces cerevisiae enol reductase OYE1, 20mM citral (citral is added in the form of a 1M substrate solution prepared with octanol, and the amount of substrate solution added Based on the amount of citral, the final concentration in the reaction system is 20 mM), and 50 mM PIPES buffer (pH 7.0) is used as the reaction medium; the concentrations all refer to the final concentration in the assay system. The reaction was carried out at 30°C, and NADH was added last, and the enzyme activity was determined by detecting the change of the absorbance value of the reaction system at 340 nm per minute (the molar coefficient of NADH ε 340 =6.3mM -1 cm -1 ). Activity unit (U) was defined as the amount of enzyme required to consume 1 μmol NADH per minute.

Embodiment 3

[0047] Example 3: Characterization of catalytic properties of Saccharomyces cerevisiae enol reductase OYE1

[0048] The total volume of the standard catalytic system is 10mL, including 20mM citral respectively (citral is added in the form of a 1M substrate solution prepared with octanol, the amount of substrate solution added is based on the amount of citral, and the final concentration in the reaction system is 20mM) , 0.25mM NAD+ , 100mM sodium formate, 0.3U / mL OYE1, 0.5U / mL FDHCB, with 50mM PIPES buffer (pH 7.0) as the reaction medium. The condition optimization of the asymmetric reduction reaction was carried out on a water-bath shaker with a rotation speed of 200rpm. The reaction solution was extracted with ethyl acetate, and the obtained organic phase was dried over anhydrous sodium sulfate, and the substrate citral and the product (R)-citronellal were quantitatively determined by gas chromatography.

[0049] The gas chromatography (Agilent 6890N) detection conditions o...

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Abstract

An enzymatic citral asymmetric reduction method capable of increasing optical purity of (R)-citronellal is disclosed, namely a method coupling an amino-acid-catalyzed citral cis-trans isomerization reaction and a citral asymmetric hydrogenation reaction catalyzed by a saccharomyces cerevisiae enol reductase OYE1 to increase the optical purity of the (R)-citronellal that is a product of citral hydrogenation. When citral cis-trans isomers are subjected to the asymmetric hydrogenation reaction catalyzed by the saccharomyces cerevisiae enol reductase OYE1 to synthesize the (R)-citronellal, the (R)-citronellal is derived from trans-citral, (S)-citronellal is derived from cis-citral, and the catalysis speed for the trans-citral is higher than that for the cis-citral. Through coupling with the amino-acid-catalyzed citral cis-trans isomerization reaction, a part of the cis-citral is converted into the trans-citral, thus greatly increasing the ee value of the product that is the (R)-citronellal. In a catalytic system having a volume of 10 mL, 100 mg / mL of glycine is added, after 50 mM citral is subjected to a catalytic reaction for 4 h, the ee value of the (R)-citronellal is 65.4%, and is increased by 48.7% when being compared with the ee value of (R)-citronellal when the cis-trans isomerization reaction is not coupled.

Description

[0001] (1) Technical field [0002] The invention relates to a method for synthesizing (R)-citronellal by catalyzing the asymmetric hydrogenation of citral by enol reductase, in particular to a cis-trans isomerization of citral through enzymatic asymmetric hydrogenation of citral and amino acid catalysis A method for improving the optical purity of the hydrogenation product (R)-citronellal by coupling reaction phases. [0003] (2) Background technology [0004] Citral belongs to the terpene fatty aldehydes, the molecular formula is C 10 h 16 O, with a strong lemon aroma, is a spice with broad application prospects. At present, the industrial production of citral is mainly chemical method, including dehydrolinalool transposition method and prenyl alcohol and prenyl aldehyde condensation rearrangement synthesis method. Citral, whether extracted from natural products or chemically synthesized, has two stereoisomers: cis-citral (neral) and trans-citral (geranial). Amino acids c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/24
CPCC12P7/24
Inventor 应向贤汪钊孟淑敏胡宝军程峰
Owner ZHEJIANG UNIV OF TECH
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