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Nano polymer-double-enzyme compound and preparation and application thereof in synthesis of (R)-2-hydroxy-4-phenyl ethyl butyrate

A technology of nano-polymers and composites, applied in the direction of nanotechnology, nanotechnology, biochemical equipment and methods, etc., can solve the problems of little practical value, high price, and low resolution yield of chloramphenicol intermediates

Active Publication Date: 2021-03-02
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical resolution method is to resolve its corresponding racemate 2-hydroxy-4-phenylbutyric acid, and then esterify it into R-HPBE. The resolution agent used is a chiral organic amine compound. The chemical resolution method exists Problem has: make resolving agent with optically pure phenylethylamine derivatives, because its price is expensive, practical value is little; And chloramphenicol intermediate resolution yield is lower (less than 50%)
Although the oil-water two-phase system has many advantages, there are also negative effects on the activity and stability of the organic relative enzyme, so people look forward to improving the activity and stability of the enzyme through the molecular modification of the enzyme

Method used

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  • Nano polymer-double-enzyme compound and preparation and application thereof in synthesis of (R)-2-hydroxy-4-phenyl ethyl butyrate
  • Nano polymer-double-enzyme compound and preparation and application thereof in synthesis of (R)-2-hydroxy-4-phenyl ethyl butyrate
  • Nano polymer-double-enzyme compound and preparation and application thereof in synthesis of (R)-2-hydroxy-4-phenyl ethyl butyrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Sequential Synthesis of Nanopolymer-Double Enzyme Complex 2-S

[0040] Synthetic route reference figure 1 In A, specifically:

[0041] (1) PAA solution: Accurately weigh 0.8mg polyacrylic acid (PAA, Mv=450000g mol -1 ), was dissolved in 40 μL distilled water to prepare 0.02 g / mL PAA stock solution (40 μL), and the pH was adjusted to 7. Mix 40 μL of PAA stock solution with 40 μL of PB buffer to obtain 80 μL of 0.01 g / mL PAA solution.

[0042] (2) Mix 80 μL of the above 0.01 g / mL PAA solution, 0.124 mg of CBR and 0.089 mg of GDH in a shaker at 30° C. and 200 rpm for 30 min to obtain a PAA mixed enzyme solution.

[0043] (3) Add 2.585 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to the PAA mixed enzyme solution in step (2), and then add PB buffer to make the solution total Volume 1mL, cross-linking at 30°C and 200rpm for 30min, add the crosslinking mixture into 100mL PB buffer, use 25kDa dialysis membrane for 3 times, 5h each time, to remove un...

Embodiment 2

[0044] Embodiment 2: Parallel synthesis of nanometer macromolecule-double enzyme complex 2-P

[0045] refer to figure 1 In B, use the parallel method to synthesize the nano-polymer-bienzyme complex 2-P, specifically:

[0046] (1) CBR-PAA conjugate: Mix 20 μL, 0.02 g / mL PAA stock solution (preparation method as shown in Example 1) with an equal volume of PB buffer, add 1.292 mg of EDC, and incubate at 30 °C, After mixing and stirring in a shaker at 200rpm for 30min, add 50μL of CBR solution (made by dissolving 0.124mg of CBR in PB buffer) dropwise, and stir for 2h, then add 50mL of PB buffer, and use a 25kDa dialysis membrane for dialysis 3 times, 5h each time , and the retentate was taken to obtain 1.8 mg of CBR-PAA conjugate.

[0047] (2) GDH-PAA conjugate: replace CBR in the CBR solution in step (1) with 0.089 mg of GDH, and perform the same operations to obtain 1.70 mg of GDH-PAA conjugate.

[0048] (3) Nanopolymer-double enzyme complex: mix 1.8mg of CBR-PAA conjugate an...

Embodiment 3

[0049] Embodiment 3: Influence of substrate concentration on reduction reaction in single aqueous phase

[0050] The two BECs (3mg 2-S or 5.5mg 2-P) prepared in Examples 1 and 2 were added to the mixture containing final concentration of 0.05mM NADP, final concentration of 0.1M d-glucose, and final concentrations of 388mM, 436mM, 485mM, 533mM, 582mM and 630mM OPBE in PB buffer, the total volume of the reaction system was 12mL. 35 DEG C, reduction reaction 40h in the constant temperature shaker of 180rpm, after the reaction finishes, add equal volume of ethyl acetate to the reaction liquid for extraction, the extract (upper layer) is dried with anhydrous sodium sulfate, and gas chromatography detects the obtained product situation, The results are shown in Table 2.

[0051] A Shimadzu GC-2014 gas chromatograph was used to analyze the substrate conversion rate and the product R-HPBE enantiomeric excess value, and the conversion rate was defined as the ratio of the converted sub...

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Abstract

The invention discloses a nano polymer-double-enzyme compound and preparation and application thereof in synthesis of (R)-2-hydroxy-4-phenyl ethyl butyrate, glucose dehydrogenase and carbonyl reductase from Saccharomyces cerevisiae CGMCC No.3361 are used as double enzymes, polyacrylic acid is used as a carrier, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is used as a cross-linking agent, a phosphate buffer solution is used as a reaction medium, mixing and stirred for 10-60 minutes in a shaking table at the temperature of 20-40 DEG C and the rotating speed of 180-220 rpm to obtain the nano polymer-double-enzyme compound. The polymer-double-enzyme compound is used for asymmetric reduction reaction of carbonyl compounds in oil and water phases, coenzyme regeneration can be realized at thesame time, and the method can improve the stability of the catalyst, prolong the service life of the catalyst, realize reutilization and improve the production efficiency.

Description

[0001] (1) Technical field [0002] The present invention relates to the preparation of ethyl (R)-2-hydroxy-4-phenylbutyrate, in particular to the use of polyacrylic acid macromolecule as a carrier to co-immobilize glucose dehydrogenase and glucose dehydrogenase derived from Saccharomyces cerevisiae CGMCCNo.3361 Carbonyl reductase, and use it for the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate to prepare ethyl (R)-2-hydroxy-4-phenylbutyrate. [0003] (2) Background technology [0004] (R)-2-hydroxy-4-phenylbutyrate (Ethyl(R)-2-hydroxy-4-phenylbutyrate, R-HPBE), CAS No. 90315-82-5, molecular formula C 12 h 16 o 3 , molecular weight 208.25, density 1.075g / mL, boiling point 212°C, insoluble in water, easily soluble in organic solvents. R-HPBE is a key chiral intermediate in the synthesis of pril drugs, and it plays an irreplaceable role in the synthesis of angiotensin-converting enzyme inhibitors (ACEI). Due to the importance of R-HPBE in the production of puril drugs...

Claims

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Application Information

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IPC IPC(8): C12N11/087C12N9/04C12P7/62C12P41/00B82Y40/00B82Y30/00
CPCC12N11/087C12N9/0006C12P7/62C12P41/002C12Y101/01184C12Y101/01119B82Y40/00B82Y30/00
Inventor 欧志敏卢媛程朋朋代洪倩王金美张楚玥唐岚
Owner ZHEJIANG UNIV OF TECH
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