A kind of sucrose isomerase mutant and its application
A sucrose isomerase and mutant technology, applied in the field of enzyme engineering, can solve the problems of increased difficulty in downstream process development, high preparation cost, inability to large-scale application, etc., and achieves the improvement of immobilized enzyme activity and stability, and the final product. The effect of high concentration and improved thermal stability
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0039] Example 1: Construction of a prokaryotic expression strain of sucrose isomerase derived from Pantoea spp.
[0040] Download the amino acid sequence of sucrose isomerase (SIM) derived from Pantoea disperse from GenBank (SEQ ID NO.1 in this paper, corresponding to GenBank accession number: AAP57083.1), remove the 1-21 signal peptide (MFLNGFKTVIALTMASSFYLA) and provide it to Beijing Qing Science Biotechnology Co., Ltd. performed the total gene synthesis of the encoding nucleic acid (using E. coli preferred codons). The C-terminal of the synthetic gene has a His tag (the pET30a(+) vector has its own His tag), and was constructed into the prokaryotic expression vector pET30a(+). The prokaryotic expression vector restriction site: 5' end Nde I, 3' end Xho I. Pass the constructed plasmid pET30a(+)-SIM through CaCl 2 Transform into Escherichia coli expression strain BL21(DE3) by heat shock transformation, spread on LB solid medium plate containing 50 μg / ml Kanamycin, cultivat...
Embodiment 2
[0042] Example 2: Purification and immobilization of sucrose isomerase derived from Pantoea disperse
[0043]Utilize the His tag carried in the SIM recombinant protein, use the activated IDA resin (purchased from Anoron (Beijing) Biotechnology Co., Ltd., specific model: His.Bind Resin, Ni-charged) to the fermentation broth obtained in Example 1 For protein purification, the specific method is as follows: 4 °C, 10000r / min, centrifuge the fermentation broth for 10min, discard the supernatant, collect the cells, wash the cells twice with phosphate buffer (pH 7.5, 0.1mol / L), and centrifuge Then, the cells were concentrated 5 times and resuspended in 20 ml of phosphate buffer (pH 7.5, 0.1 mol / L). The bacterial solution after the above treatment was placed in ice water for ultrasonic fragmentation until clarification. The ultrasonic fragmentation conditions were: working for 2s, interval of 5s, and ultrasonic power of 500W. The fragmented lysate was centrifuged in a low-temperature...
Embodiment 3
[0047] Example 3: Construction of SIM prokaryotic expression strain E. coli BL21(DE3) / pET30a(+)-SIM error-prone mutation library
[0048] Using pET30a(+)-SIM recombinant plasmid as PCR template, conventional T7F / R as universal primer (primer sequence: T7F:5'-TAATACGACTCACTATAGGG-3'T7R:GCTAGTTATTGCTCAGCGG see SEQ ID NO.3 and 4) to carry out the SIM gene. Error-prone PCR amplification, adjust Mg in PCR amplification reaction system 2+ , Mn 2+ , dCTP and dTTP oligonucleotide concentrations, so that the base mismatch rate of the mutant library is only 2 / 1000, that is, to ensure that only 1 to 2 amino acids are mutated in a mutant.
[0049] Error-prone PCR reaction system:
[0050]
[0051] Error-prone PCR reaction conditions: pre-denaturation at 95°C for 5 min; then denaturation at 94°C for 30 s, annealing at 56°C for 1 min, extension at 72°C for 1.5 min, a total of 25 cycles; and finally extension at 72°C for 10 min.
[0052] 2 μL of the above error-prone PCR product was sa...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More