D-阿洛酮糖 3-差向异构酶(D-psicose 3-epimerase,DPE)可以催化D-果糖转化为稀有糖D-阿洛酮糖,是生产D-阿洛酮糖过程中的关键酶。为了提高DPE的表达水平,该研究以枯草芽孢杆菌(Bacillus subtilis) WB800为宿主菌,异源表达Clostridium scindens ATCC 35704来源的DPE。首先,构建不同单启动子和串联启动子介导DPE表达的重组菌,通过摇瓶培养,发现由单启动子Phag介导的重组菌株经发酵后酶活力最高,最高酶活力为19.62 U/mL,是P43介导的原始菌株酶活力的1.3倍。最后对Phag的核糖体结合位点(ribosome binding site,RBS)序列进行突变,突变后的菌株酶活力再次提高了29.4%,是原始菌株酶活力的1.69倍。该研究结果为工业化生产DPE提供了方法学参考。
D-psicose 3-epimerase can catalyze the conversion of D-fructose to the rare sugar D-psicose, which is the key enzyme in the production of D-psicose. In order to increase the expression level of D-psicose 3-epimerase, B. subtilis WB800 was used as the host strain to heterologously express D-psicose 3-epimerase from Clostridium scindens ATCC 35704. First, recombinant strains with different single promoters and tandem promoters were successfully obtained. Through culturing in shaking flasks, the recombinant strains with the single promoter Phag showed the highest enzyme activity (approximately 19.62 U/mL), which was 1.3 times that of the original strain with P43. Then the Phag′s ribosome binding site sequence was mutated, and the enzyme activity of the mutant strain was further increased by 29.4%, which was 1.69 times that of the original strain. The research results provide a methodological reference for the industrial production of D-psicose 3-epimerase.
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