Sucrose isomerase mutant, coding gene and application of sucrose isomerase mutant
A technology of sucrose isomerase and coding gene, which is applied in the preparation of isomaltulose from sucrose by sucrose isomerase mutant biocatalysis, the field of sucrose isomerase mutant and its coding gene can solve the problem of staying in the laboratory and other issues to achieve the effect of improving the catalytic efficiency
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Embodiment 1
[0025] Example 1: Cloning and expression of sucrose isomerase gene in Escherichia coli E. coli BL21 (DE3).
[0026] Construction of the expression vector: the synthesis of the sucrose isomerase gene described below was completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The sucrose isomerase gene (GenBank: G37835) derived from Erwinia sp. Ejp617 was seamlessly cloned into pET28b(+) vector between Nco I and Xho I by PCR to obtain the sucrose isomerase gene Expression vector pET28b(+)-SI( Figure 4). The operation of the PCR program was as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 s, annealing at 53-58°C for 15 s, extension at 72°C for 1.5 minutes, a total of 25 cycles; and extension at 72°C for 10 minutes.
[0027] The preparation method of competent cells is as follows: obtain the E.coli BL21 (DE3) strain preserved in a glycerol tube from a -80°C refrigerator, streak on an anti-antibiotic LB plate, culture at 37°C for 10 hours, and ob...
Embodiment 2
[0029] Example 2: Induced expression and purification of sucrose isomerase recombinant bacteria
[0030] Wet bacteria containing sucrose isomerase: Inoculate the recombinant E. coli BL21(DE3) / pET28b(+)-SI obtained in Example 2 into LB liquid culture containing 50 μg / mL kanamycin resistance cultured at 37°C and 200rpm for 12h, then inoculated with 1% (v / v) inoculum into fresh LB liquid medium containing 50μg / mL kanamycin resistance, and cultivated at 37°C at 150rpm until Cell OD 600 After reaching 0.6-0.8, add IPTG with a final concentration of 0.1mM, induce culture at 25°C for 10h, centrifuge at 8000rpm at 4°C for 20min, discard the supernatant, collect the precipitate, and wash twice with pH 7.0, 50mM Tris HCl buffer , that is, to obtain the wet cells of the recombinant strain E.coli BL21(DE3) / pET28b(+)-SI containing sucrose isomerase; add the wet cells to pH 7.0, 50mM Tris HCl buffer to resuspend, in ice water The mixture was subjected to ultrasonic crushing for 20 min, an...
Embodiment 3
[0032] Embodiment 3: Construction and screening of sucrose isomerase gene library.
[0033] (1) Introducing beneficial mutations by error-prone PCR
[0034] Plasmid DNA was isolated from E. coli BL21(DE3) / pET28b(+)-SI recombinant E. coli with MiniBEST Plasmid Purification Kit (TaKaRa, Dalian, China). In containing 60ng plasmid (pET28b-SI) as template, 60pmol each mutagenic primer (E2-F, E2-R, Table S1), 0.4mM MnCl 2 , 2x in 50 μL reaction mixture for amplification of DNA fragments TaqPCR StarMix and Loding Dye (TaKaRa, Dalian, China). The amplification process of the gene encoding SIase was as follows: 94°C for 2 minutes, followed by 30 cycles of 94°C for 30s, 60°C for 30s and 72°C for 2 minutes, and finally placed at 72°C for 10 minutes. This protocol resulted in an average mutation frequency of every 1000bp base substitution. The PCR product was digested using QuickCut Dpn I at 37°C for 3 hours to remove the template. Then, library gene fragments were purified using Mi...
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