D-N-carbamoylase and application thereof
A technology of carbamyl hydrolase and carbamyl, which is applied in the field of D-N-carbamyl tryptophan and its application, can solve the problems of low catalytic activity and poor solubility of D-N-carbamyl tryptophan, and achieve good solubility, The effect of mild reaction conditions and good application and development prospects
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Embodiment 1
[0032] Example 1 Cloning of D-carbamyl hydrolase gene
[0033] The D-N-carbamyl hydrolase gene in the above-mentioned Arthrobacter was cloned by a one-step cloning method.
[0034] (1) First, the above-mentioned Arthrobacter was cultured overnight at 37° C. using LB medium. After the bacterial cells were obtained by centrifugation, the total genomic DNA was obtained using a conventional bacterial genome extraction kit.
[0035] (2) Design degenerate primers according to the reported D-N-carbamyl hydrolase gene (upstream primer: CGC GGATTC WTGSSSAAWWACTTR, downstream primers: CGCGAATTCTYAGTCWTTSASKTT), using the Arthrobacter genome as a template for PCR, the system is as follows (μL): 10×PCR Mix 10, upstream primer 0.2, downstream primer 0.2, genome 0.2, DNA polymerase 0.2, ddH 2 O 9.2. The PCR program was: pre-denaturation at 95°C for 10 min, cleavage at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 29 cycles, and extension at 72°C for 10 min. Th...
Embodiment 2
[0036]Example 2 Construction and cultivation of recombinant Escherichia coli BL21(DE3) / pET28a-AshyuC
[0037] Plasmids pET28a and AshycC were double digested with restriction endonucleases BamH I and EcoR I in a water bath at 37°C overnight, purified by agarose gel electrophoresis the next day, and the target fragments were recovered using an agarose recovery kit. At 37°C, the gene AshyuC was ligated with the digested plasmid pET28 using T4 DNA ligase to obtain the recombinant expression vector pET28a-AshyuC( figure 2 ). Heat the constructed recombinant expression vector pET28a-AshyuC into Escherichia coli BL21(DE3) competent, coat a solid plate containing kanamycin-resistant LB, and perform colony PCR verification after overnight culture. The positive clone is the recombinant E. coli BL21(DE3) / pET28a-AshyuC. Pick positive clones and culture them overnight in LB medium, then transfer them into fresh LB culture at 2% transfer amount the next day, and culture them to OD 600 ...
Embodiment 3
[0038] Example 3 Separation and purification of D-carbamyl hydrolase
[0039] Suspend the recombinant cells in solution A (20mmol·L -1 Sodium phosphate, 500mmol·L -1 NaCl, 20mmol·L -1 imidazole, pH 7.4), the crude enzyme solution was obtained after sonication and centrifugation. The column used for purification is an affinity column HisTrap FF crude (nickel column), which is accomplished by using the histidine tag on the recombinant protein for affinity binding. First, use solution A to equilibrate the nickel column, load the crude enzyme solution, continue to use solution A to elute the breakthrough peak, and after equilibrium, use solution B (20mmol L -1 Sodium phosphate, 500mmol·L -1 NaCl, 1000mmol·L -1 imidazole, pH 7.4) for gradient elution to elute the recombinant protein bound to the nickel column to obtain recombinant D-N-carbamoylase. Enzyme activity assay (DL-N-carbamoyl tryptophan as substrate) and SDS-PAGE analysis ( image 3 ). Depend on image 3 It can b...
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