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Method for synthesizing (S)-citronellol through double-enzyme coupling

A technology of citronellol and old yellow enzyme, which is applied in the field of biocatalysis, can solve the problems of reduced atom economy, lack of high activity, and can not meet the requirements of practical application, and achieves superior site selectivity and high atom economy. Effect

Pending Publication Date: 2022-01-14
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of special enzymes with high activity, the current biological method can not meet the requirements of practical application.
In addition, the biological reduction of nerol, citronellal or citral requires the configuration of a coenzyme cycle system. In order to drive the coenzyme cycle, it is often necessary to add an excess of cosubstrates, thus reducing the atom economy

Method used

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  • Method for synthesizing (S)-citronellol through double-enzyme coupling
  • Method for synthesizing (S)-citronellol through double-enzyme coupling
  • Method for synthesizing (S)-citronellol through double-enzyme coupling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Preparation of wet bacteria and crude enzyme solution expressing alcohol dehydrogenase YsADH and old yellow enzyme NemR-PS

[0035] 1. Construction of Alcohol Dehydrogenase YsADH Genetic Engineering Bacteria

[0036] The artificially synthesized disclosed source is from Yorkella sp.WZY002; preserved in the China Type Culture Collection Center, address: Wuhan, China, Wuhan University, postcode: 430072, preservation number: CCTCC No: M2013099, preservation date: 2013 On March 22, the gene encoding alcohol dehydrogenase YsADH was disclosed in the patent application CN201310188883.9), and its amino acid sequence and nucleotide sequence are respectively shown in SEQ ID NO.1 and SEQ ID NO.2.

[0037] The construction of genetically engineered bacteria E.coli BL21(DE3) / pET-28b-YsADH: Insert the gene encoding alcohol dehydrogenase YsADH shown in SEQ ID NO.2 into the Nco I and Hind III restriction enzyme sites of the pET28b vector, The recombinant vector pET28b-YsADH...

Embodiment 2

[0059] Example 2: Construction of initial reaction system for synthesizing (S)-citronellol based on coenzyme self-cycle with nerol as substrate

[0060] The wet thallus of alcohol dehydrogenase YsADH prepared in step 3 of Example 1 and the wet thallus of old yellow enzyme NemR-PS are mixed in a buffer (50mM Tris-HCl) in a certain proportion as a catalyst, and nerolidol is used as a substrate. Add coenzyme NADP + , constituting a total reaction system of 10 mL. The substrate nerolidol is preformed into a 1M solution with ethanol as the solvent, and then added appropriately according to the final concentration of the substrate in the reaction system.

[0061] The initial reaction system without condition optimization is as follows: the final concentration of substrate nerol is 60mM, the coenzyme NADP + The final concentration is 0.4mM, the final concentration of wet cells is 120g / L (the wet cells of alcohol dehydrogenase YsADH and the wet cells of old yellow enzyme NemR-PS are...

Embodiment 3

[0066] Embodiment 3: Taking nerol as the substrate based on the optimum temperature of coenzyme self-circulation synthesis (S)-citronellol

[0067] Select 25°C, 30°C, 35°C, 40°C, 45°C, 50°C and 55°C, and other operations and reaction conditions are the same as in Example 2. Three sets of parallel experiments were performed each time, and the average value and standard error were calculated. The results were as follows: Figure 5 shown. At 25°C, the conversion rate of (S)-citronellol was 43.75%; when the catalytic temperature was 40°C, the conversion rate had reached 72.39%. When the temperature increased to 45 and 50 °C, the conversion decreased to 65.78% and 55.27%. In summary, the optimum reaction temperature is 40°C.

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Abstract

The invention discloses a method for synthesizing (S)-citronellol through double-enzyme coupling. The method comprises the following steps of mixing wet bacteria obtained by respectively fermenting and culturing an engineering bacterium containing an alcohol dehydrogenase YsADH gene and an engineering bacterium containing an old yellow enzyme NemR-PS gene as a catalyst, taking nerol as a substrate, taking NADP <+> as a coenzyme, and taking a buffer solution with the pH value of 6-9 as a reaction medium to form a reaction system; and after reaction is completed under the conditions of 25-55 DEG C and 0-900rpm, separating and purifying reaction liquid to finally obtain the (S)-citronellol. When 100 mM nerol is used as the substrate, the conversion rate of the product (S)-citronellol after 12 hours of reaction is as high as 99.74%, and the e.e. value of the product is greater than 99%. Compared with the prior art, the established method for synthesizing the (S)-citronellol is green and efficient, is high in atom economy, and has excellent site selectivity, chemical selectivity and enantiomer selectivity.

Description

(1) Technical field [0001] The invention belongs to the field of biocatalysis, and relates to a method for synthesizing (S)-citronellol based on coenzyme self-circulation with nerol as a substrate. (2) Background technology [0002] Citronellol has a rose aroma and is an important ingredient in perfumes. The fragrance type of (S)-citronellol is different from that of (R)-citronellol, which can meet the preparation requirements of high-end perfumes. (S)-citronellol has a variety of biological activities, and can inhibit the activity of Salmonella typhi and Staphylococcus aureus. (S)-citronellol is an important intermediate in the synthesis of chiral chemicals, and can be used to synthesize all-cis rose ether, (S)-pulegone and 3(S)-methyl-heptanoic acid, etc. [0003] The chemical synthesis of (S)-citronellol usually takes nerol, citronellal, citral, etc. as substrates, and utilizes chemical hydrogenation to obtain (S)-citronellol. Citronellal and citral have both C=C and C...

Claims

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Application Information

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IPC IPC(8): C12P7/04C12N15/70C12N9/02C12N9/04C12R1/19
CPCC12P7/04C12N9/0006C12N9/0036C12N15/70C12Y106/99001
Inventor 应向贤邵帅王崎舟
Owner ZHEJIANG UNIV OF TECH
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