Enzyme-catalyzed method for synthesizing (2S, 3R)-2-substituted aminomethyl-3-hydroxybutyrate

Patent metadata

Field	Value
Publication number	US-11802299-B2
Application number	US-202117545963-A
Country	US
Kind code	B2
Filing date	Dec 8, 2021
Priority date	Jun 8, 2021
Publication date	Oct 31, 2023
Grant date	Oct 31, 2023

Abstract

An enzyme-catalyzed method of synthesizing (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate, including: preparing engineered bacteria containing a carbonyl reductase SsCR-encoding gene; preparing a resting cell suspension of the engineered bacteria; preparing a culture containing carbonyl reductase; and mixing the culture containing carbonyl reductase with substrate 2-substituted aminomethyl-3-one butyrate, glucose dehydrogenase, a cosolvent, glucose and a cofactor followed by asymmetric carbonyl reduction to obtain (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate. The amino acid sequence of the carbonyl reductase is shown in SEQ ID NO.1.

First claim

What is claimed is:

1. A method of synthesizing (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate, comprising:
(1) preparing engineered bacteria containing a a carbonyl reductase SsCR-encoding gene;
(2) preparing a resting cell suspension of the engineered bacteria;
(3) inoculating the resting cell suspension of the engineered bacteria into a kanamycin-containing medium to prepare a supernatant containing carbonyl reductase SsCR; and
(4) mixing the supernatant with 2-substituted aminomethyl-3-one butyrate (II), glucose dehydrogenase, a cosolvent, a phosphate-buffered saline, glucose and a cofactor to obtain a reaction mixture; and subjecting the reaction mixture to an asymmetric carbonyl reduction to obtain the (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate (I), as shown in the following reaction scheme:

wherein R 1 is selected from the group consisting of hydrogen, tert-butoxycarbonyl, benzyloxycarbonyl, trityl, formyl, trifluoroacetyl, benzoyl, mono-substituted benzoyl, poly-substituted benzoyl and heteroaryl formyl; and R 2 is selected from the group consisting of C 1 -C 10 alkyl, cycloalkyl, phenethyl, substituted phenethyl, phenyl and substituted phenyl; and
the carbonyl reductase SsCR comprises the amino acid sequence of SEQ ID NO:1.

2. The method of claim 1 , wherein the engineered bacteria contain a plasmid pET28a-SsCR, and a host cell is Escherichia coli BL21 (DE3).

3. The method of claim 2 , wherein the supernatant is prepared through steps of:
inoculating the resting cell suspension of the engineered bacteria into the kanamycin-containing medium followed by activation on a shaker; when an OD 600 value reaches 0.6-1.2, adding an inducer followed by continuous culture and centrifugation to collect a cell precipitate; suspending the cell precipitate with a buffered solution to obtain a cell suspension with a cell concentration of 1-20 g/100 mL; and subjecting the cell suspension to ultrasonic or compressing disruption followed by centrifugation to obtain the supernatant containing the carbonyl reductase SsCR.

4. The method of claim 3 , wherein the inducer is isopropyl β-D-thiogalactoside (IPTG) with a concentration of 0.05-0.8 mM.

5. The method of claim 4 , wherein after the inducer is added, the culture is performed at 15° C.-25° C. for 8 h-24 h.

6. The method of claim 3 , wherein the buffered solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate solution; and a concentration of the buffered solution is 30 mM-300 mM.

7. The method of claim 1 , wherein in the asymmetric carbonyl reduction, a concentration of the 2-substituted aminomethyl-3-one butyrate (II) is 5-300 g/L; a concentration of the carbonyl reductase SsCR in the supernatant is 1%-20%; a concentration of the glucose dehydrogenase is 0.1-1 g/L; a concentration of the glucose is 1-600 g/L; and a concentration of the cofactor is 0.01-0.5 mM.

8. The method of claim 7 , wherein the asymmetric carbonyl reduction is performed at 20° C.-40° C.; and a pH of the phosphate buffered saline is 5.0-8.0.

9. The method of claim 8 , wherein in the asymmetric carbonyl reduction, the cosolvent is selected from the group consisting of dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, n-hexane, cyclohexane, acetonitrile, ethyl acetate, dichloromethane, acetone, 1,2-dichloroethane, methanol, ethanol, isopropanol and a combination thereof.

10. The method of claim 9 , wherein in the asymmetric carbonyl reduction, after the 2-substituted aminomethyl-3-one butyrate (II) is monitored by supercritical fluid chromatography (SFC) to be completely consumed, the reaction mixture is subjected to extraction 3-4 times with an equal volume of ethyl acetate, and organic phases are collected, combined, washed twice with saturated sodium chloride, dried with anhydrous sodium sulfate, and subjected to vacuum distillation to remove ethyl acetate to obtain a target product (2S,3R)-2-substituted aminomethyl-3-hydroxybutyrate (I).

Assignees

• Univ Fudan

Inventors

• Chen Fener
• Tao Yuan
• Huang Zedu
• Cheng Dang
• Meng Ge

Classifications

• C12P13/001 Primary

  Amines; Imines · CPC title

• C12Y101/01184

  Carbonyl reductase (NADPH) (1.1.1.184) · CPC title

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12P41/002

  by oxidation/reduction reactions · CPC title