Organophosphorus hydrolases and applications thereof
A technology of organophosphorus and hydrolase, applied in the direction of hydrolase, enzyme, enzyme, etc., can solve the problems of low yield, low enzyme activity, complicated process, etc., achieve high activity of degrading organophosphorus, and improve the effect of degradation activity
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Embodiment 1
[0026] Obtaining mutants and fusions of engineered organophosphate degrading enzymes
[0027] Wild-type organophosphate hydrolase (Organophosphorus hydrolase, OPH, GenBank accession number M29593, gene name opd) was selected from the nucleic acid database. In order to improve the degradation activity of the most widely used organophosphate pesticides, such as chlorpyrifos, etc., 8 beneficial mutation sites of wild-type OPH were integrated, as shown in Table 1, and a new modified organophosphate hydrolase was rationally designed. MOPD1, its amino acid sequence is shown in SEQ ID NO.1, and its coding gene is named mopd1. The N-terminal of the ice nucleation protein derived from Pseudomonas bacteria (the gene name is inaQN, the protein name is InaQN) is selected as the anchor protein, and its corresponding amino acid sequence is shown in SEQ ID NO.3. InaQN is fused with MOPD1 to obtain organophosphate hydrolase MOPD2, and its corresponding amino acid sequence is shown as SEQ ID ...
Embodiment 2
[0034] Construction of Recombinant Expression Vector of Organophosphate Degrading Enzyme Gene
[0035] The vector pMOPD was used as the basic skeleton, and the plasmid sequence was shown in SEQ ID NO.11, and the recombinant expression vectors pMOPD1, pMOPD2, pMOPD3, and pMOPD4 were respectively constructed. Specific steps are as follows:
[0036] (1) Construction of recombinant expression vectors pMOPD1 and pMOPD4
[0037] The mopd1 gene fragment containing the corresponding restriction site sequence was digested with FastDigest endonucleases Bgl II and Kpn I. The reaction system was: 5 μL 10*FD buffer, 2.5 μL Bgl II, 2.5 μL Kpn I, 30 μL containing the corresponding enzymes mopd1 gene fragment of cut site sequence and 10 μL ultrapure water. The reaction conditions are: 37°C, 2h. The mopd1 gene fragment which was double digested with Bgl II and Kpn I was obtained by purifying and recovering with the kit. The mopd1 gene fragment digested with Bgl II and Kpn I was connected t...
Embodiment 3
[0048] Acquisition of Recombinant Escherichia coli Cells and Degradation Activity of Organophosphate Hydrolase
[0049] 1. Acquisition of recombinant E. coli cells
[0050] The constructed vectors pMOPD1, pMOPD2, pMOPD3, and pMOPD4 were transformed into host cells E. coli SyBE_002444 by electric shock respectively, and recombinant strains OPE1, OPE2, OPE3, and OPE4 were respectively obtained.
[0051] 2. Production of organophosphate hydrolase
[0052] Recombinant strains OPE1, OPE2, OPE3 and OPE4 were cultured to the logarithmic phase in LB medium at 37°C, and then transferred to M9 medium containing glucose for fermentation. The fermentation temperature was 30°C and the shaker speed was 200rpm. The target When protein reaches the maximum production amount, finish fermentation to obtain fermented liquid. Take 10ml of the fermentation broth, centrifuge, harvest the cells, resuspend in 5mL of PBS buffer solution with pH=7.4, ultrasonically break and centrifuge at 12000r / min f...
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