Double enzyme tandem preparation method of l-2-aminobutyric acid
Disclosed is a double enzyme tandem preparation method of L-2-aminobutyric acid, and belongs to the field of bioengineering. In the disclosure, recombinant Escherichia coli expressing L-glutamate mutase and recombinant Escherichia coli expressing L-aspartate-β-decarboxylase are separately cultured to obtain L-glutamate mutase and L-aspartate-β-decarboxylase. The two enzymes are added to a reaction system at a certain mass ratio, and L-glutamate is used as a substrate to carry out an enzyme reaction to prepare the L-2-aminobutyric acid. When the dosage of the L-aspartate-β-decarboxylase is 2 mg/mL, and the reaction time is 24 h, 8.5 mmol/L L-2-aminobutyric acid is produced by conversion, with a molar conversion rate of 85.00%. Compared with a chemical production method, the method disclosed by the disclosure has a safe production process and no environmental pollution. Compared with a multi-enzyme synthesis system with threonine as a substrate, the substrate is cheaper and the process is simpler.
1. A method for biosynthesizing L-2-aminobutyric acid, comprising using L-glutamate as a substrate and a double enzyme tandem reaction system to catalyze L-glutamate to produce L-2-aminobutyric acid, wherein the two enzymes in the double enzyme tandem reaction system are L-glutamate mutase and L-aspartate-B-decarboxylase, wherein the amino acid sequence of the L-aspartate-B-decarboxylase is set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4; in the double enzyme tandem reaction system, a concentration of the substrate L-glutamate is 10-30 mmol/L; the L-glutamate mutase and the L-aspartate-B-decarboxylase are added to the double enzyme tandem reaction system at a ratio of (4-6):1; and a dosage of the L-aspartate-B-decarboxylase is 0.5-5 mg/mL.
2. The method of claim 1 , wherein in the double enzyme tandem reaction system, the concentration of the substrate L-glutamate is 10-20 mmol/L, and the L-glutamate mutase and the L-aspartate-β-decarboxylase are added to the double enzyme tandem reaction system at a ratio of (4-6):1.
3. The method of claim 1 , wherein the dosage of the L-aspartate-β-decarboxylase is 2-3 mg/mL.
4. The method of claim 1 , wherein the double enzyme tandem reaction system also contains 0.5-1.5 mmol/L dithiothreitol, 0.5-1 mmol/L pyridoxal phosphate, 0.01-0.02 mmol/L adenosylcobalamin, 0.02-0.2 mmol/L pyruvic acid and 18-22 mM K 2 HPO 4 /KH 2 PO 4 buffer at a pH of 6.5-7.5.
5. The method of claim 1 , wherein a temperature of a catalytic reaction is 35-39° C., and the reaction time is 10-30 h.
6. The method of claim 1 , wherein the nucleotide sequence of the gene encoding the L-glutamate mutase is set forth in SEQ ID NO:5.
7. The method of claim 1 , wherein a preparation method of the L-glutamate mutase comprises: ligating the gene encoding L-glutamate mutase and having the nucleotide sequence set forth in SEQ ID NO:5 to a plasmid pET-28a to obtain a recombinant plasmid pET-28a-GlmES, transforming the recombinant plasmid into Escherichia coli BL21 to obtain recombinant Escherichia coli BL21/pET-28a-GlmES, and fermenting the recombinant Escherichia coli BL21/pET-28a-GlmES to produce the L-glutamate mutase.
8. The method of claim 7 , wherein the fermentation is culturing the Escherichia coli in an environment containing IPTG, and induce culturing at 28-32° C. for 15-30 h.
9. The method of claim 8 , wherein the L-glutamate mutase is prepared by: collecting and breaking the cultured bacterial cells, and separating and purifying a target enzyme protein to obtain an electrophoretically pure enzyme.
10. The method of claim 1 , wherein the L-aspartate-β-decarboxylase is prepared by: ligating the gene encoding the L-aspartate-β-decarboxylase and having the nucleotide sequence as set forth in any of SEQ ID NO:6 to SEQ ID NO:9 to the plasmid pET-28a to obtain a recombinant plasmid, transforming the recombinant plasmid into Escherichia coli BL21 to obtain recombinant Escherichia coli , and fermenting the recombinant Escherichia coli to produce the L-aspartate-β-decarboxylase.
11. The method of claim 9 , wherein the fermentation is culturing the Escherichia coli in an environment containing IPTG, and induce culturing at 28-32° C. for 15-30 h.
12. The method of claim 11 , wherein the preparation method of the L-aspartate-β-decarboxylase further comprises: collecting and breaking the cultured bacterial cells, and separating and purifying a target enzyme protein to obtain an electrophoretically pure enzyme.