Magnetic immobilization of yeast cells and its application in the preparation of ethyl (r)-2-hydroxy-4-phenylbutyrate

Abstract

The invention discloses a magnetically immobilized yeast cell and its application in the preparation of (R)-2-hydroxy-4-phenylbutyrate ethyl ester. The magnetically immobilized yeast cell is made of lysine-modified magnetic nano The particles are mixed with the yeast cell solution, fixed in a shaker at 20°C-40°C, 130-170r / min for 2-6h, after the reaction, magnetically separated, the precipitate is washed with deionized water, and freeze-dried to obtain magnetically immobilized yeast cell. The immobilization method of the invention effectively improves the stability of the yeast cells and enhances the tolerance of the cells to the substrate solution. Under the action of an external magnetic field, the magnetically immobilized cells are easier to separate from the reaction system, which is beneficial to the separation and extraction of products and simplifies the process flow. Appropriate magnetic field frequency and magnetic field strength in the alternating magnetic field reactor can effectively enhance the mass transfer process of the reaction system, significantly shorten the reduction reaction time, and then improve the biotransformation efficiency.

Description

(1) Technical field

[0001] The present invention relates to the preparation of ethyl (R)-2-hydroxy-4-phenylbutyrate, in particular to the use of amino acid-modified magnetic nanoparticles as a carrier to effectively immobilize yeast and use it for 2-oxo Preparation of (R)-2-hydroxy-4-phenylbutyric acid ethyl ester by asymmetric reduction of ethyl 4-phenylbutyrate. (2) Background technology

[0002] (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 an important chiral intermediate for the synthesis of lisinopril, quinapril, ramipril, cilazapril and other angiotensin-converting enzyme inhibitors (ACEI) antihypertensive and congestive heart failure drugs R-HPBE can be prepared by asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE).

[0003] Hypertension is...

Examples

Embodiment 1

[0027] Embodiment 1: magnetic nanometer iron tetroxide (Fe 3 o 4 ) preparation

[0028] Weigh 12.675g FeCl 2 Dissolved in 1000mL deionized water, configured as 0.1M FeCl 2 solution. Weigh 16.220g FeCl 3 Dissolved in 1000mL deionized water, configured as 0.1M FeCl 3 solution. FeCl 2 Solution 30mL, FeCl 3 Mix 60mL of the solution into a 250mL three-neck flask, add 20mL of deionized water, and stir at a high speed of 1000 rpm, keep a water bath at 30-40°C, slowly add ammonia water dropwise to adjust the pH to 10, and the solution turns black and shiny. Raise the temperature to 60°C and react for 1h. Then raise the temperature to 80°C and ripen for 1h. After the reaction was finished, it was cooled to room temperature, and it was repeatedly washed with deionized water until it was colorless and transparent, and the pH of the supernatant was neutral, and collected by an external magnetic field to obtain a black magnetic particle precipitate (Fe 3 o 4 ), pre-frozen in -8...

Embodiment 2

[0029] Embodiment 2: the magnetic nano ferric oxide (Fe) of amino acid modification 3 o 4 - Preparation of AA)

[0030] Accurately weigh 0.2g each of the four amino acids (lysine, arginine, glutamic acid, and aspartic acid) and dissolve them in 200mL of deionized water. After shaking well, add 0.2g of magnetic Fe 3 o 4 After the nanoparticles were shaken properly, they reacted under the action of 40KHz ultrasonic wave for half an hour, took out the Erlenmeyer flask, collected the black precipitate with an external magnetic field, washed it with deionized water for 3 times, and then put it in a -80°C refrigerator for 8 hours. into a freeze dryer for 12 hours to obtain amino acid-modified Fe 3 o 4 Nanoparticles (denoted as Fe 3 o 4 -AA) 0.2-0.4 g. Among them, the lysine-modified magnetic nano-ferric iron tetroxide (Fe 3 o 4 -Lys) 0.35g, Fe 3 o 4 -Arg 0.27g, Fe 3 o 4 -Glu 0.23g, Fe 3 o 4 -Asp 0.21g. Identification of Fe by Fourier Transform Infrared Transform Spec...

Embodiment 3

[0031] Embodiment 3: the screening of active dry yeast

[0032] Weigh 3g of the purchased 11 kinds of active dry yeast and add them to 11 Erlenmeyer flasks filled with 20mL phosphate buffer (pH 7), shake well, and after fully dissolved, add them to the 11 kinds of 20mL yeast cell solutions respectively. 1mL of 0.2mmol / mL OPBE ethanol solution was placed in a shaker at 30°C and 180r / min for 24h. After the reaction was completed, the transformation solution was taken out and centrifuged at 8000rpm for 10min. The supernatant was collected and washed with an equal volume of Extracted with ethyl acetate, naturally volatilized and concentrated to a volume of 3 mL to make a sample to be tested by gas phase, the results are shown in Table 1, and No. 7 active yeast was selected for subsequent tests, that is, Baizuan high-activity dry yeast of Angel Yeast Co., Ltd. ( high sugar tolerance).

[0033] Gas chromatography (GC) detection conditions: chiral chromatographic column CP7502 (25m×...

(1) Technical field

[0001] The present invention relates to the preparation of ethyl (R)-2-hydroxy-4-phenylbutyrate, in particular to the use of amino acid-modified magnetic nanoparticles as a carrier to effectively immobilize yeast and use it for 2-oxo Preparation of (R)-2-hydroxy-4-phenylbutyric acid ethyl ester by asymmetric reduction of ethyl 4-phenylbutyrate. (2) Background technology

[0002] (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 an important chiral intermediate for the synthesis of lisinopril, quinapril, ramipril, cilazapril and other angiotensin-converting enzyme inhibitors (ACEI) antihypertensive and congestive heart failure drugs R-HPBE can be prepared by asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE).

[0003] Hypertension is...

Examples

Embodiment 1

[0027] Embodiment 1: magnetic nanometer iron tetroxide (Fe 3 o 4 ) preparation

[0028] Weigh 12.675g FeCl 2 Dissolved in 1000mL deionized water, configured as 0.1M FeCl 2 solution. Weigh 16.220g FeCl 3 Dissolved in 1000mL deionized water, configured as 0.1M FeCl 3 solution. FeCl 2 Solution 30mL, FeCl 3 Mix 60mL of the solution into a 250mL three-neck flask, add 20mL of deionized water, and stir at a high speed of 1000 rpm, keep a water bath at 30-40°C, slowly add ammonia water dropwise to adjust the pH to 10, and the solution turns black and shiny. Raise the temperature to 60°C and react for 1h. Then raise the temperature to 80°C and ripen for 1h. After the reaction was finished, it was cooled to room temperature, and it was repeatedly washed with deionized water until it was colorless and transparent, and the pH of the supernatant was neutral, and collected by an external magnetic field to obtain a black magnetic particle precipitate (Fe 3 o 4 ), pre-frozen in -8...

Embodiment 2

[0029] Embodiment 2: the magnetic nano ferric oxide (Fe) of amino acid modification 3 o 4 - Preparation of AA)

[0030] Accurately weigh 0.2g each of the four amino acids (lysine, arginine, glutamic acid, and aspartic acid) and dissolve them in 200mL of deionized water. After shaking well, add 0.2g of magnetic Fe 3 o 4 After the nanoparticles were shaken properly, they reacted under the action of 40KHz ultrasonic wave for half an hour, took out the Erlenmeyer flask, collected the black precipitate with an external magnetic field, washed it with deionized water for 3 times, and then put it in a -80°C refrigerator for 8 hours. into a freeze dryer for 12 hours to obtain amino acid-modified Fe 3 o 4 Nanoparticles (denoted as Fe 3 o 4 -AA) 0.2-0.4 g. Among them, the lysine-modified magnetic nano-ferric iron tetroxide (Fe 3 o 4 -Lys) 0.35g, Fe 3 o 4 -Arg 0.27g, Fe 3 o 4 -Glu 0.23g, Fe 3 o 4 -Asp 0.21g. Identification of Fe by Fourier Transform Infrared Transform Spec...

Embodiment 3

[0031] Embodiment 3: the screening of active dry yeast

[0032] Weigh 3g of the purchased 11 kinds of active dry yeast and add them to 11 Erlenmeyer flasks filled with 20mL phosphate buffer (pH 7), shake well, and after fully dissolved, add them to the 11 kinds of 20mL yeast cell solutions respectively. 1mL of 0.2mmol / mL OPBE ethanol solution was placed in a shaker at 30°C and 180r / min for 24h. After the reaction was completed, the transformation solution was taken out and centrifuged at 8000rpm for 10min. The supernatant was collected and washed with an equal volume of Extracted with ethyl acetate, naturally volatilized and concentrated to a volume of 3 mL to make a sample to be tested by gas phase, the results are shown in Table 1, and No. 7 active yeast was selected for subsequent tests, that is, Baizuan high-activity dry yeast of Angel Yeast Co., Ltd. ( high sugar tolerance).

[0033] Gas chromatography (GC) detection conditions: chiral chromatographic column CP7502 (25m×...

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