Sucrose isomerase mutant with high activity and high conversion rate and application thereof
A technology of sucrose isomerase and high conversion rate, which is applied in the direction of isomerase, application, transferase, etc., can solve the problems of low enzyme utilization rate, limited pace, inevitable by-products, etc., and achieve the improvement of activity and conversion rate , Improve production efficiency, broaden the effect of actual production and application prospects
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Embodiment 1
[0040] A sucrose isomerase mutant Y246L with high activity and high conversion rate, the specific steps of its preparation method are as follows:
[0041] (1) Use the vector pET-28a(+)-pal-2 carrying the sucrose isomerase gene as a template to carry out site-directed mutation to construct the mutant plasmid pET-28a(+)-Y246L, and the mutation primers are as follows (the underline is the mutation point) :
[0042] The forward mutation primer is shown in SEQ ID NO.9, wherein CG TTG CG, the underline is the mutant base;
[0043] The reverse mutation primer is shown in SEQ ID NO.10, wherein GC AAC GC, the underline is the mutant base.
[0044] (2) Perform PCR amplification under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 5 min, 35 cycles, and finally incubation at 68°C for 10 min. PCR amplified products were detected by agarose gel electrophoresis, recovered and purified by ta...
Embodiment 2
[0050] A sucrose isomerase mutant H287R with high activity and high conversion rate, the specific steps of its preparation method are as follows:
[0051] (1) Use the vector pET-28a(+)-pal-2 carrying the sucrose isomerase gene as a template to carry out site-directed mutation to construct the mutant plasmid pET-28a(+)-H287R, and the mutation primers are as follows (the underline is the mutation point) :
[0052] The forward mutation primer is shown in SEQ ID NO.11, wherein GT CGC GT, the underline is the mutant base;
[0053] The reverse mutation primer is shown in SEQ ID NO.12, wherein AC GCG AC, underlined are mutant bases.
[0054] (2) Perform PCR amplification under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 5 min, 35 cycles, and finally incubation at 68°C for 10 min. PCR amplified products were detected by agarose gel electrophoresis, recovered and purified by tappin...
Embodiment 3
[0060] A sucrose isomerase mutant H481P with high activity and high conversion rate, the specific steps of its preparation method are as follows:
[0061] (1) Use the vector pET-28a(+)-pal-2 carrying the sucrose isomerase gene as a template to carry out site-directed mutation to construct the mutant plasmid pET-28a(+)-H481P, and the mutation primers are as follows (the underline is the mutation point) :
[0062] The forward mutation primer is shown in SEQ ID NO.13, wherein TC CCG GT, the underline is the mutant base;
[0063] The reverse mutation primer is shown in SEQ ID NO.14, wherein AG GGC CA, the underline is the mutant base.
[0064] (2) Perform PCR amplification under the following conditions: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 1 min, extension at 68°C for 5 min, 35 cycles, and finally incubation at 68°C for 10 min. PCR amplified products were detected by agarose gel electrophoresis, recovered and purified by t...
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