A Bacillus subtilis engineered bacterium capable of highly expressing glucose dehydrogenase
A technology of glucose dehydrogenase and Bacillus subtilis, which is applied in the field of bioengineering and genetic engineering, can solve the problems of poor thermal stability, low expression, interference with the sensitivity of blood glucose detection electrodes, etc., and achieve the effect of increasing production and reducing inhibition
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[0057] Preparation of seed solution: Pick a single colony of the recombinant bacterium B. subtilis from the Kana-resistant solid medium, inoculate it into LB liquid medium, and culture it on a shaking table at 37°C and 200r / min for 8h.
[0058] Shake flask fermentation culture: 4% volume fraction of the seed solution was transferred to the fermentation medium, 30°C, 220r / min, and cultured for 48h.
[0059] The detection method involved in the following examples is:
[0060] Determination of bacterial biomass: Take 20mL of bacterial liquid with different concentrations, centrifuge at 8000r / min for 10min, wash twice with PB buffer solution with pH 7.0 and 50mmol / L, measure the wet weight, dry at 105°C to a constant value, weigh dry weight.
[0061] The wet weight of the bacteria is about 3.8 times the dry weight.
[0062] Determination of glucose dehydrogenase enzyme activity: Take 100 μL of appropriately diluted enzyme solution, add 3 mL of chromogenic solution (50 mmol / L, PB...
Embodiment 1
[0071] Embodiment 1: Construction of recombinant plasmid
[0072] The FAD-glucose dehydrogenase (FAD-GDH) gene derived from Burkholderia cepacia (Burkholderia cepacia) on pUC57 / gdh was amplified by PCR (see Table 1 for primers), and the amplification The product was recovered, and double-digested by BamH Ⅰ and Mlu Ⅰ, and inserted into the same double-digested plasmid pMA5 with the promoter P HpaⅡ Downstream, construct the recombinant plasmid pMA5 / gdh (the construction process is as follows figure 1 ).
[0073] The obtained recombinant plasmids were respectively transformed into E.coli JM109, screened on the ampicillin resistance plate, the plasmids were extracted from the positive transformants, and verified by double-enzyme electrophoresis with BamH Ⅰ and Mlu Ⅰ respectively (the verification results are as follows: figure 2 ), the gel electrophoresis band is consistent with the size of the target gene, and the correct gene sequencing will be verified and compared with the ...
Embodiment 2
[0074] Embodiment 2: the construction of recombinant bacterium
[0075] Transfer the recombinant plasmid pMA5 / gdh obtained in Example 1 into B. subtilis competent WB600, select positive clones from the plate containing Kanna resistance, and obtain the recombinant strain B. subtilis / pMA5 / gdh to obtain the recombinant bacterial shake flask Ferment and cultivate for 48 hours to obtain a fermentation liquid, collect the bacteria by centrifugation, break the cells of the bacteria, and centrifuge to obtain a supernatant, which is the enzyme solution.
[0076] The obtained enzyme solution was tested for enzyme activity, and the test result was: the intracellular enzyme activity of the recombinant strain B.subtilis / pMA5 / gdh was 962U / L. Take 30 μL of the enzyme solution obtained from B.subtilis / pMA5 / gdh, add 10 μL of 4× loading buffer, boil for 10 minutes, and perform SDS-PAGE detection. The results are shown in image 3 , because there is also a 60kDa band in the blank control, so th...
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