Glucose isomerase mutant and application thereof
A technology for glucose isomerase and mutants, which is applied in the field of glucose isomerase mutants, can solve the problem that the thermostability of high-temperature-resistant enzymes does not reach industrialized production and the like, and achieves the effect of excellent reusability.
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Embodiment 1
[0023] Example 1: Construction and screening of ToGI single point mutants
[0024] 1. Mutant construction
[0025] According to the parental sequence of ToGI (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2), the mutation primers for site-directed mutation were designed, and the recombinant vector pET28b / ToGI was used as a template by using rapid PCR technology. To introduce a single mutation at position 216, the primers are:
[0026] Forward primer GAACCCA NNN TTCGCTCACG (the underline is the mutated base)
[0027] reverse primer GAGCGAA NNN TGGGTTCAGAC (underline is the mutant base)
[0028] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.
[0029] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 57°C for 6.5min) for 30 cycles; 72...
Embodiment 2
[0040] Example 2: Construction and screening of two-site mutants of glucose isomerase
[0041] According to the sequence of the single mutant ToGI-I constructed in Example 1, the mutation primers for site-directed mutation were designed, and using the rapid PCR technology, the recombinant vector pET28b / ToGI-I was used as a template to introduce a single mutation at position 228. The primers were:
[0042] Forward primer GAACTTC NNN CACGCTGTTG (the underline is the mutated base)
[0043] reverse primer CAGCGTG NNN GAAGTTCAGAC (the underline is the mutant base)
[0044] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.
[0045] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 57°C for 6.5min) for 30 cycles; 72°C for 5min.
[0046] The PCR product was transformed into E.coliBL21 (DE3) co...
Embodiment 3
[0053] Example 3: Construction and screening of three-site mutants of glucose isomerase
[0054] Design mutation primers for site-directed mutation according to the mutant ToGI-II sequence constructed in Example 2, and use the rapid PCR technology to use the recombinant vector pET28b / ToGI-II as a template to introduce a single mutation at position 345. The primers are:
[0055] Forward primer GCTGGGT NNN TACTCTCGTG (the underline is the mutated base)
[0056] reverse primer GAGAGTA NNN ACCCAGCAGAC (the underline is the mutated base)
[0057] PCR reaction system: 2×Phanta Max Buffer (containing Mg 2+ ) 25μL, dNTPs 10mM, forward primer 2μL, reverse primer 2μL, template DNA 1μL, Phanta Max Super-Fidelity DNA Polymerase 50U, add ddH 2 0 to 50 μL.
[0058] The PCR amplification conditions were 95°C for 3min; (95°C for 15s, 50°C for 15s, 60°C for 6.5min) 30 cycles; 72°C for 5min.
[0059] The PCR product was transformed into E.coliBL21 (DE3) competent cells, and a single clo...
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