High-temperature resistance glucose isomerase, its coding gene and use
A technology of glucose isomerase and coding gene, which is applied in high temperature-resistant glucose isomerase and its coding gene and application field, and achieves the effect of good heat resistance
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[0073] 1) Gene amplification and plasmid construction
[0074] Design primers according to gene bank (L38994) gene sequence T1: 5'AGA TTA CCT AGG TACATA TGG CTG AAT TCT ATC CAG AAA TCC CGA AA 3' and T2: 5'ATT TCC GATGGC GCG CCT CAC CTC AGT TCC AGA ATG GTC TTC 3 '; The gene encoding glucose isomerase (TN) was amplified from Thermotoga neapolitana using primer pair T1 and T2.
[0075] Amplification reaction conditions were: 5 microliters of 10× buffer (Promega, USA), 4 microliters of 2.5 millimolar dNTPs (Promega, USA), 2 microliters of 10 micromolar primer T1, 2 microliters of 10 micromolar primer T2, 0.5 microliter 5U / microliter Tag DNA polymerase (Promega, USA), pick a little Thermotoga neapolitana bacteria with an inoculation loop, and then use sterile water to adjust the reaction volume to 50 microliters.
[0076] The PCR amplification reaction program was: 95°C for 3 minutes, 40 cycles: 95°C for 50 seconds, 50°C for 30 seconds, 72°C for 1 minute, and finally 72°C for 10 m...
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