Mutant microorganism and method for producing peptide using the same
A technology for microbial and peptide synthesis, applied in biochemical equipment and methods, microorganisms, microorganisms, etc., can solve the problems of slow synthesis speed and low recovery rate of peptides
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Embodiment 1
[0116] Determination of the aspartylphenylalanine decomposition activity of each peptidase gene deletion strain of embodiment 1
[0117] In the operation shown below, Escherichia coli ATCC8739 is used as the parent strain, and the aminoacyl histidine dipeptidase (pepD), isoaspartyl dipeptidase (idaA), and α-aspartyl dipeptidase genes of the parent strain (pepE gene) and leucyl aminopeptidase (pepA) were sequentially disrupted, and the pepD gene deletion strain, pepD iadA double deletion strain, pepD iadA pepE triple deletion strain, and pepD iadA pepE pepA quadruple deletion strain were constructed, and their days were measured. Partylphenylalanine decomposing activity.
[0118] (1) Deletion of aminoacylhistidine dipeptidase gene (pepD gene)
[0119] Synthesize primers based on the sequence information of the pepD gene in the base sequence database (DDBJ / EMBL / GeneBank registration index number is AE000132)
[0120] 5'-GATCTGGCGCACTAAAAACC (sequence number 1)
[0121] 5'-GGG...
Embodiment 2
[0145] Determination of the aspartylphenylalanine decomposition activity of each peptidase gene deletion strain of embodiment 2
[0146] The parental strain and each gene-deficient strain were inoculated with 4ml LB medium respectively, and cultured with shaking at 30°C for 16 hours. Centrifuge 4ml of the culture at 2200×g for 15 minutes to obtain bacterial pellets. Add 4 ml of physiological saline to the cell pellet, and centrifuge at 2200×g for 15 minutes to obtain washed cells. Suspend the washed cells in 1 ml of sonication solution (20 mM MOPS-KOH buffer (pH 7.0) containing 1 mM DTT). The cell suspension was ultrasonically disrupted (produced by Bioruptor, 7.5 minutes), and then centrifuged at 12000×g for 10 minutes to obtain a cell-free extract. Protein was quantified by the Bradford method using a protein analysis CBB solution (manufactured by Nacalai Tesque). Bovine serum albumin from Sigma was used as the standard protein.
[0147] Add 100 μl of cell-free extract t...
Embodiment 3
[0150] The determination of the aspartyl phenylalanine decomposition activity of each peptidase gene deletion strain of embodiment 3
[0151] The parental strain and each gene-deleted strain were inoculated in 3ml LB (1.0% tryptone from Bacto company, 1.0% yeast extract from Bacto company, 1.0% sodium chloride, pH 7.0) culture medium, placed at 37°C and shaken for 16 hours. Take 1ml of the culture and centrifuge at 2200×g for 15 minutes to obtain bacterial pellet. Add 1 ml of physiological saline to the cell pellet, and centrifuge at 2200×g for 15 minutes to obtain washed cells. Add 0.1 ml of substrate solution (100 mM alanyl glutamine, 100 mM boric acid-NaOH buffer solution, pH 9.0) to the washed cells, and react at 30° C. for 6 hours.
[0152] Alanylglutamine in the reaction solution was quantified by HPLC under the following conditions to determine the amount of decomposition of alanylglutamine. Column: Inertsil ODS-2 (4.6×250mm, GL Science Company). Mobile phase: 5mM so...
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