A heat-resistant lyase mmppgh and polynucleotide encoding the enzyme
A polynucleotide, lyase technology, applied in the directions of lyase, botanical equipment and methods, biochemical equipment and methods, etc.
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Embodiment 1
[0017] Example 1: Cloning and expression of thermostable lyase MMPpgh
[0018] 1. Amplification of the lyase gene, (with Meiothermus TG17 phage MMP17 genomic DNA as template)
[0019] (1) The primer sequences used for the amplification of the thermophilic bacteriophage MMP17 lyase MMPpgh gene are as follows:
[0020] Forward primer: 5'- CCG GAATTC ATGCGCATCGTTCATCCC -3'
[0021] Reverse primer: 5'- CCC AAGCTT TTGCAATGCGCGATTTGT -3';
[0022] (2) The amplification system is as follows:
[0023] Table 1: Amplification reaction system
[0024] ;
[0025] (3) The amplification conditions are as follows:
[0026] Mix the reaction system evenly, pre-denature at 95°C for 3 min, then denature at 95°C for 40 s, anneal at 58°C for 30 s, extend at 72°C for 40 s, and after 30 cycles, extend at 72°C for 8 min. After the reaction, take 3 μL of the product and perform electrophoresis analysis in 1% agarose gel (see figure 1 ), the size of the MMPpgh gene PCR product was 633bp...
Embodiment 2
[0069] Embodiment 2: Partial enzymatic properties and enzymatic activity experiments of thermostable lyase MMPpgh
[0070] 1. Effect of thermostable lyase MMPpgh on the optimum temperature, pH value and metal ions on thermostable lyase MMPpgh
[0071] In order to study the optimum action temperature of lyase MMPpgh, the influence of pH value and metal ions on the lyase, the TG17 bacterial liquid cultivated to the logarithmic phase was used; the TG17 bacterial liquid in the logarithmic growth phase was centrifuged at 4500rpm for 20min, and the supernatant was discarded . After the precipitate was suspended in 10 mL of PBS (pH 7.4), it was ultrasonically broken and centrifuged to obtain the precipitate, which was re-suspended in 5 mL of PBS. At this time, the TG17 cell wall is all cell fragments, which serve as the reaction substrate of the cleavage enzyme MMPpgh. The initial OD of the TG17 reaction substrate was measured 600 Value = 0.241.
[0072] (1) Optimum catalytic tem...
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