Exoinulinase Z2-5 with low-temperature activity and gene of exoinulinase Z2-5
A technology of exo-inulinase activity, applied in the field of genetic engineering, can solve the problem of less exo-inulinase and achieve good application potential
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Embodiment 1
[0034] Example 1: Cloning of Sphingobacterium exo-inulinase Z2-5
[0035] Extraction of Sphingobacterium genomic DNA: Centrifuge the bacterium cultured in liquid for 2 days to get the bacteria, add 1mL lysozyme, treat at 37°C for 60min, then add the lysate, lyse in a water bath at 70°C for 60min, mix once every 10min, in 4 Centrifuge at 10000 rpm for 5 min. The supernatant was extracted in phenol / chloroform to remove impurity proteins, and then an equal volume of isopropanol was added to the supernatant. After standing at room temperature for 5 minutes, centrifuge at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, the precipitate was washed twice with 70% ethanol, dried in vacuo, dissolved by adding an appropriate amount of TE, and stored at -20°C for later use.
[0036] The degenerate primers Inu32F and Inu32R were designed and synthesized according to the conserved amino acid sequence of exo-inulinase (H-N-W-M-N-D-P-N-G and R-D-P-K-V-F-W-H-E-Q-S) (Table 1)....
Embodiment 2
[0041] Embodiment 2: Preparation of recombinant exo-inulinase Z2-5
[0042] Using Z2-5InuEF and Z2-5InuER as a primer pair (Table 1) and Sphingobacterium genomic DNA as a template, PCR amplification was performed. The PCR reaction parameters were: denaturation at 94°C for 5 min; then denaturation at 94°C for 30 sec, annealing at 45°C for 30 sec, extension at 72°C for 2 min, and after 40 cycles, incubation at 72°C for 5 min and incubation at 25°C for 3 min. The nucleotide sequence of the mature peptide encoded by the exo-inulinase gene Z2-5 was obtained from the PCR results. Ligate the nucleotide sequence encoding the mature peptide of the exo-inulinase gene Z2-5 with the expression vector pEASY-E1 to obtain the recombinant plasmid pEASY-E1-Z2-5 containing the exo-inulinase gene Z2-5 And transform Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli strain BL21(DE3) / Z2-5.
[0043] Take the E. coli BL21 (DE3) strain containing the recombinant plasmid pEASY-E1-Z2-5...
Embodiment 3
[0044] Example 3: Characterization of Purified Recombinant Exo-Inulinase Z2-5
[0045] 1. Activity analysis of recombinant exo-inulinase Z2-5
[0046] The activity determination method of the purified recombinant exo-inulinase Z2-5 in Example 2 adopts the 3,5-dinitrosalicylic acid (DNS) method: the substrate is dissolved in 0.1M buffer solution, and its final concentration is 0.5% (w / v); the reaction system contains 100μL appropriate amount of enzyme solution and 900μL substrate; after the substrate is preheated at the reaction temperature for 5 minutes, add the enzyme solution and react for 10 minutes, then add 1.5mL DNS to terminate the reaction, and boil for 5 minutes , after cooling to room temperature, measure the OD value at a wavelength of 540nm. One enzyme activity unit (U) is defined as the amount of enzyme required to decompose the substrate to produce 1 μmol of reducing sugar per minute under the given conditions.
[0047] 2. Determination of optimum pH and pH sta...
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