The enzyme activity and stability of recombinant human ADH1B and ALDH2 expressed in Bacillus subtilis
Objective To obtain active and stable expression of recombinant human alcohol dehydrogenase 1B(ADH1B)and aldehyde dehydrogenase 2(ALDH2)from Bacillus subtilis(B.subtilis).Methods Encoding sequencing of human ADH1B and ALDH2 were obtained and cloned into expression vector pBE2R.The recombinant plasmids pBE2R-adh1B and pBE2R-aldh2 were constructed and transformed into B.subtilis WB600 using the chemical transformation method.Recombinant B.subtilis WB600 was cultured at 37℃,200 r/min for 24 h;the expression of recombinant human ADH1B and ALDH2 were confirmed by Western blotting with the specific antibodies.The enzyme activities were confirmed by classic method using specific kits.The stability of the recombinant B.subtilis WB600 strains was analyzed by treatment with artificial gastric fluid in vitro.Results After 24 hours of incubation at 37℃and 200 r/min,recombinant human ADH1B and ALDH2 were successfully identified in the supernatant of the culture medium and the bacterial sediment.The enzyme activities of recombinant human ADH1B and ALDH2 in the supernatant were respectively 3.67 U/mL and 1.61 U/mL.The amounts of recombinant strains after treatment with artificial gastric fluid under pH 2,3,or 4 for different time were close to those before treatment.Conclusion Recombinant human ADH1B and ALDH2 were successfully expressed in B.subtilis WB600 with enzyme activities and the recombinant B.subtilis WB600 strains could tolerate the strong acid environment.
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