A starch branching enzyme mutant with improved catalytic ability
A technology of starch branching enzymes and mutants, applied in the field of enzyme engineering
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Embodiment 1
[0035] Embodiment 1: the preparation of starch branching enzyme mutant
[0036] The starch branching enzyme derived from Rhodothermus obamensis STB05 and the branching enzyme derived from Escherichia coli combined with maltoheptaose (cited from the literature: Feng L, Fawaz R, Hovde S, et al.Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides[ J].Biochemistry,2015,54(40):6207-18.) crystal structure overlap, using the latter as a template, by comparing the distance between the amino acid of the substrate binding site and the substrate, respectively selected For the far 158 position and the 489 position which is relatively close, the R158T (amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.9) and Q489E mutations were designed body (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO.10).
[0037] ...
Embodiment 2
[0042] Embodiment 2: Contain the construction of the genetically engineered bacteria expressing the starch branching enzyme mutant gene of the present invention
[0043] At 37°C, treat the PCR product with DpnI for 2 hours, then transform the processed PCR product into E.coli JM 109, spread the transformed E.coli JM 109 on LB agar containing 100 μg / mL ampicillin cultured overnight in a 37°C incubator for 12 hours, from which a single colony was selected and inoculated into LB liquid medium containing 100 μg / mL ampicillin, cultured overnight at 37°C, 200 r / min and extracted according to the plasmid extraction kit Plasmids were extracted, identified and sequenced according to the method indicated in the manual. The constructed target plasmid was transformed into the competent expression host E.coli BL21(DE3) by chemical transformation. Finally, the genetically engineered bacteria E.coli BL21(DE3)(pET-20b(+) / gbe) was obtained.
Embodiment 3
[0044] Embodiment 3: the expression of starch branching enzyme mutant of the present invention
[0045] Activation culture of host bacteria: E.coli BL21(DE3)(pET-20b(+) / gbe) was streaked and separated on LB solid medium, placed in a constant temperature incubator at 37°C for overnight culture, and positive single colonies were picked Inoculate in a 250mL Erlenmeyer flask containing 50mL LB liquid medium. Place the centrifuge tube in a rotary shaker at 200 r / min, and incubate at 37° C. for 8-10 h.
[0046] Fermentation culture: Inoculate 1 mL of the activated strain culture solution into a 250 mL Erlenmeyer flask containing 50 mL of TB liquid medium, place it in a rotary shaker at a rate of 200 r / min, and culture it at 37°C for 96 hours.
[0047] Ampicillin was added to each medium at a final concentration of 100 μg / mL before use.
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