Method for producing 1, 2-alkamine compound by utilizing whole-cell transformation

Abstract

The invention discloses a method for producing a 1, 2-alkamine compound by utilizing whole-cell transformation, and belongs to the technical field of gene engineering and microbiology engineering. A bacillus subtilis engineering strain co-expresses alcohol dehydrogenase (MnADH) and w-transaminase (PAKw-TA), and can whole-cell catalyze phenyl-1, 2-ethylene glycol at one step so as to synthesize 2-amino-1-phenyethyl alcohol. By utilizing the method provided by the invention, the phenyl-1, 2-ethylene glycol can be used as a substrate, and whole-cell transformation is carried out to prepare the high-purity 2-amino-1-phenyethyl alcohol; and the method for producing the 1, 2-alkamine compound by utilizing whole-cell transformation is convenient to operate, cheap in substrate, and not only beneficial to improving the transformation efficiency but also beneficial to reducing the reaction cost, and has an important industrial application value.

Description

Technical field

[0001] The invention relates to a method for producing 1,2-amino alcohol compounds by whole cell transformation, and belongs to the technical fields of genetic engineering and microbial engineering. Background technique

[0002] 2-Amino-1-phenylethanol is an important pharmaceutical intermediate and can be used as a synthetic raw material for Metoprolol and Nebivolol for the treatment of cardiovascular diseases. Therefore, 2-amino-1-phenylethanol has important applications in the fields of medicine and chemical synthesis.

[0003] At present, the preparation of 2-amino-1-phenylethanol is mainly through chemical synthesis and biological transformation. Among them, although the process of chemical synthesis is mature, the reaction conditions are severe and many by-products are generated. Sometimes it is necessary to use some organic solvents that pollute the environment; in contrast, the conditions of the biotransformation method are relatively mild, safe and The co...

Examples

Embodiment 1

[0044] Example 1: Construction of recombinant plasmid

[0045] Specific steps are as follows:

[0046] (1) According to the mnadh gene sequence (SEQ ID NO.1) in the whole genome nucleic acid sequence of Mycobacterium neoaurum in NCBI, design PCR primers P3 and P4 (SEQ ID NO.3 and SEQ ID NO.4) of the alcohol dehydrogenase MnADH ;

[0047] (2) According to the whole genome nucleic acid sequence of Pseudomonas aeruginosa PAK in NCBI (GenBank: LR657304.1) pakω-ta gene sequence (No. 345243-346610) (coded amino acid sequence is SEQ ID NO. 2), design ω-transaminase PCR primers P5 and P6 of PAKω-TA (SEQ ID NO.5 and SEQ ID NO.6);

[0048] (3) Using the above genomic DNA as a template, using the above primers for PCR amplification, the amplification conditions are: 95°C pre-denaturation for 5 min, one cycle; 95°C denaturation, 1 min, 58°C annealing, 1 min, 72°C extension for 1 min 30 s, 30 cycles; final extension at 72°C for 10 minutes, after the amplification is completed, the PCR product is...

Embodiment 2

[0050] Example 2: Construction of recombinant bacteria

[0051] Specific steps are as follows:

[0052] Take 100μL of E.coli BL21 competent cells into a 1.5mL centrifuge tube, add 5μL of the recombinant plasmid pMA5-mnadh-pakω-ta to be transformed, gently pipette, and place on ice for 45min; place the centrifuge tube After precise heat shock at 42°C for 90s, place it on ice for 5 minutes, then add 800μL of LB liquid medium and incubate at 37°C in a shaker for 1-1.5h; after centrifugation, discard most of the supernatant, re-suspend the remaining bacteria The solution was spread on an LB plate containing ampicillin resistance, and the plasmid was extracted after the transformant grew out for verification, and the recombinant bacteria B.s168 / pMA5-mnadh-pakω-ta was obtained.

Embodiment 3

[0053] Example 3: Verification of recombinant bacteria

[0054] Specific steps are as follows:

[0055] (1) The recombinant bacteria B.s168 / pMA5-mnadh-pakω-ta obtained in Example 2 was activated with LB medium and cultured at 37° C. and 160 r / min for 12 hours to obtain seed liquid;

[0056] (2) Transfer the seed solution to 100 mL of LB broth medium with 1% inoculum, and continue to cultivate for 2 hours to OD 600 Is 0.8 to obtain the fermentation broth;

[0057] (3) After the fermentation broth was induced at 7°C for 12h, centrifuged at 4°C and 8000r / min for 10min to collect the bacteria;

[0058] (4) After washing the bacteria with pH 7.5 phosphate buffer solution twice, add the bacteria to the catalytic system and react at 37°C for 10 hours to obtain the reaction solution; among them, the OD of the bacteria in the catalytic system 600 =30, in vitro, the catalytic system also contains 100mM substrate phenyl-1,2-ethylene glycol, 5mM L-Glu, 0.02mM NADP + P and 275mM NH 4 Cl(pH 8.0);

[0...

Technical field

[0001] The invention relates to a method for producing 1,2-amino alcohol compounds by whole cell transformation, and belongs to the technical fields of genetic engineering and microbial engineering. Background technique

[0002] 2-Amino-1-phenylethanol is an important pharmaceutical intermediate and can be used as a synthetic raw material for Metoprolol and Nebivolol for the treatment of cardiovascular diseases. Therefore, 2-amino-1-phenylethanol has important applications in the fields of medicine and chemical synthesis.

[0003] At present, the preparation of 2-amino-1-phenylethanol is mainly through chemical synthesis and biological transformation. Among them, although the process of chemical synthesis is mature, the reaction conditions are severe and many by-products are generated. Sometimes it is necessary to use some organic solvents that pollute the environment; in contrast, the conditions of the biotransformation method are relatively mild, safe and The co...

Examples

Embodiment 1

[0044] Example 1: Construction of recombinant plasmid

[0045] Specific steps are as follows:

[0046] (1) According to the mnadh gene sequence (SEQ ID NO.1) in the whole genome nucleic acid sequence of Mycobacterium neoaurum in NCBI, design PCR primers P3 and P4 (SEQ ID NO.3 and SEQ ID NO.4) of the alcohol dehydrogenase MnADH ;

[0047] (2) According to the whole genome nucleic acid sequence of Pseudomonas aeruginosa PAK in NCBI (GenBank: LR657304.1) pakω-ta gene sequence (No. 345243-346610) (coded amino acid sequence is SEQ ID NO. 2), design ω-transaminase PCR primers P5 and P6 of PAKω-TA (SEQ ID NO.5 and SEQ ID NO.6);

[0048] (3) Using the above genomic DNA as a template, using the above primers for PCR amplification, the amplification conditions are: 95°C pre-denaturation for 5 min, one cycle; 95°C denaturation, 1 min, 58°C annealing, 1 min, 72°C extension for 1 min 30 s, 30 cycles; final extension at 72°C for 10 minutes, after the amplification is completed, the PCR product is...

Embodiment 2

[0050] Example 2: Construction of recombinant bacteria

[0051] Specific steps are as follows:

[0052] Take 100μL of E.coli BL21 competent cells into a 1.5mL centrifuge tube, add 5μL of the recombinant plasmid pMA5-mnadh-pakω-ta to be transformed, gently pipette, and place on ice for 45min; place the centrifuge tube After precise heat shock at 42°C for 90s, place it on ice for 5 minutes, then add 800μL of LB liquid medium and incubate at 37°C in a shaker for 1-1.5h; after centrifugation, discard most of the supernatant, re-suspend the remaining bacteria The solution was spread on an LB plate containing ampicillin resistance, and the plasmid was extracted after the transformant grew out for verification, and the recombinant bacteria B.s168 / pMA5-mnadh-pakω-ta was obtained.

Embodiment 3

[0053] Example 3: Verification of recombinant bacteria

[0054] Specific steps are as follows:

[0055] (1) The recombinant bacteria B.s168 / pMA5-mnadh-pakω-ta obtained in Example 2 was activated with LB medium and cultured at 37° C. and 160 r / min for 12 hours to obtain seed liquid;

[0056] (2) Transfer the seed solution to 100 mL of LB broth medium with 1% inoculum, and continue to cultivate for 2 hours to OD 600 Is 0.8 to obtain the fermentation broth;

[0057] (3) After the fermentation broth was induced at 7°C for 12h, centrifuged at 4°C and 8000r / min for 10min to collect the bacteria;

[0058] (4) After washing the bacteria with pH 7.5 phosphate buffer solution twice, add the bacteria to the catalytic system and react at 37°C for 10 hours to obtain the reaction solution; among them, the OD of the bacteria in the catalytic system 600 =30, in vitro, the catalytic system also contains 100mM substrate phenyl-1,2-ethylene glycol, 5mM L-Glu, 0.02mM NADP + P and 275mM NH 4 Cl(pH 8.0);

[0...