A kind of aldoxime dehydratase and its application in catalytic synthesis of aromatic nitrile compounds
A dehydratase and compound technology, applied in the field of biocatalysis research, to achieve the effect of simplifying the production process, reducing production costs and promoting upgrading
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Embodiment 1
[0035] Example 1 Construction of recombinant expression plasmid pROxdF1
[0036]The aldoxime dehydratase Oxd gene from Pseudomonas putida F1 (DSMZ 6899) was cloned using primers Oxd_UP and Oxd_down ( figure 1 ), the nucleotide sequence of the gene is shown in SEQ ID NO.1. The nucleic acid sequences of primers Oxd_UP and Oxd_down are:
[0037] 5'-CGCGGATCCGATGGAATCTGCAATCGATAAACA-3';
[0038] 5'-ATAAGAATGCGGCCGCCTAGTTGGAACTGACTTTTGC-3'.
[0039] The Oxd-F1 gene was digested with Bam HI and Not I, and the digested gene fragment was recovered; at the same time, the expression plasmid pRSFDuet-1 was digested with Bam HI and Not I, and the digested plasmid fragment was recovered. T4 ligase was used for ligation, and the ligated product was transformed into a cloned host E. coli DH5α. LB solid plate containing kanamycin resistance was used for screening, and positive transformants were selected. The sequencing result showed that the recombinant plasmid after the gene sequence ...
Embodiment 2
[0040] Example 2 Construction of genetically engineered bacteria BL21-pROxdF1 and catalyst preparation
[0041] The recombinant expression plasmid pROxdF1 constructed in Example 1 was transformed into the expression host E. coli BL21 (DE3) to obtain genetically engineered bacteria BL21-pROxdF1. BL21-pROxdF1 was inoculated into a 10 mL test tube of liquid LB medium (38 μg / mL kanamycin), and cultured with shaking at 37° C. and 200 rpm for 12-16 h. The culture solution was inoculated into 50 mL of liquid auto-induction medium (38 μg / mL kanamycin) in a 250 mL triangular conical flask according to the inoculum amount of 1 to 5%, and the culture was shaken for 2 to 3 hours at 37 ° C and 200 rpm. When strain density OD 600 When the value reached 0.6 to 0.8, the culture temperature was adjusted to 18 °C, and the culture was continued for 12 h at 200 rpm.
[0042] Cells were collected by centrifugation at 5000-10000 × g for 5-10 min, which was used as a catalyst for dehydration to sy...
Embodiment 3
[0043] Example 3 Characterization of aldoxime dehydratase OxdF1
[0044] Using 5mM 3-pyridinealdoxime as the substrate, in 0.5mL 0.1M potassium phosphate buffer (pH 6.0) reaction system, add 50μL of genetically engineered bacteria BL21-pROxdF1 resting cells, and measure its catalytic activity in the range of 20~55℃ vitality, such as image 3 As shown, the optimum reaction temperature was 35°C.
[0045] Using 5mM 3-pyridinealdoxime as the substrate, in the buffer system of different pH values (pH 4.0-9.5), add 50 μL of genetically engineered bacteria BL21-pROxdF1 resting cells, and measure the catalytic activity at 35 °C. like Figure 4 As shown, the optimum reaction pH was 7.0.
[0046] The resting cells of genetically engineered bacteria BL21-pROxdF1 were placed in a water bath at 30 and 35 °C, respectively, and their catalytic activity was measured after maintaining for different times. Figure 5 As shown, its half-life reaches about 3.8 hours at 30°C.
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