A kind of L-glufosinate-ammonium chemical enzymatic production method
A production method and chemical enzymatic technology, applied in the field of bioengineering, can solve the problems of poor substrate tolerance, low optical purity of products, difficult separation, etc., and achieve strict stereoselectivity, cheap and easily available raw materials, and high catalytic activity. Effect
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Embodiment 1
[0026] Synthesis of D,L-glufosinate-ammonium phosphono-substituents.
[0027]Take the synthetic method of N-phenylacetyl-D, L-glufosinate-ammonium as an example: add 500 mL of 4M NaOH solution to a 1000 mL four-necked flask equipped with a stirring and thermometer, and stir with 104.2 g of 95% glufosinate-ammonium (0.5 mol) until completely dissolved. Transfer the filtrate to a 1 L one-necked flask. Use a rotary evaporator to increase the vacuum in the single-necked flask to above 0.095MPa, raise the temperature, and release the ammonia gas in the reaction system. When about 50mL of water is removed, it is the end point. And add 50mL of water.
[0028] Transfer the liquid in the one-necked bottle to a four-necked bottle, and transfer it to a refrigerator, lower the temperature to 0°C, add 94.6g 98% phenylacetyl chloride (0.6mol) dropwise, the dropping time is 1-1.5 hours, and the temperature is controlled at 0 -5°C, after the dropwise addition, directly add 16g of NaOH soli...
Embodiment 2
[0036] Construction of recombinant amidase Bm-Ami mutant.
[0037] Using the reported amino acid sequence of amidase with high stereoselectivity as a probe, the potential amidase sequence was searched in Genbank according to the sequence homology. Combined with the characteristic structure, conserved sequence and catalytic triplet in the amidase tag sequence, the amidase gene Bm-ami (AF161313.1) from Bacillus megaterium was screened for artificial synthesis. The synthetic gene was connected with the expression vector pET-28a (Novagen company), and the expression recombinant plasmid pET28-Bm-ami containing the amidase gene was constructed, and transformed into competent cells E.coli BL21(DE3). It was confirmed by DNA sequencing that the nucleotide sequence of the cloned parent amidase was identical to that reported by NCBI.
[0038] In order to perform site-directed mutagenesis of the methionine (M) at the 309th position of Bm-Ami, primers M309F and M309R (see Table 2) were de...
Embodiment 3
[0044] Vitality identification of parental amidase and amidase mutant engineered bacteria.
[0045] The starting strain E.coli BL21(DE3) / pET28-Bm-ami, mutant strain E.coli BL21(DE3) / pET28-M309V, E.coli BL21(DE3) / pET28-M309V / F319Y and E.coli BL21( DE3) / pET28-M309V / F319Y / Y442F were respectively inoculated into LB liquid medium containing 50mg / L kanamycin and cultured at 37°C for 12h, and then inoculated with 1% inoculation amount (v / v) into fresh LB liquid medium containing 50mg / L kanamycin In the LB liquid medium of L Kanamycin, cultivate to the cell concentration OD 600 About 0.6, then add IPTG with a final concentration of 0.1mM to the LB liquid medium, induce culture at 28°C for 10h, centrifuge at 8000g for 10min at 4°C, and collect the bacterial cells containing the recombinant amidase.
[0046] Reaction system composition: 100mM racemic N-phenylacetyl-D, L-glufosinate-ammonium, Tris-HCl (pH 8.5, 100mM) and a certain amount of cells, reacted at 30°C, 150r / min for 0.5h, too...
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