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文章摘要
L-鼠李树胶糖激酶在D-阿洛酮糖合成中的应用
Application of L-rhamnulose Kinase on Production of D-psicose
投稿时间:2019-04-17  修订日期:2019-05-23
DOI:
中文关键词: D-阿洛酮糖  L-鼠李树胶糖激酶  D-阿洛酮糖-3-差向异构酶  多聚磷酸盐激酶
英文关键词: D-psicose  L-rhamnulose kinase  D-psicose-3-epimerase  polyphosphate kinase
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目)
作者单位E-mail
温俊婷 江南大学生物工程学院 1547480332@qq.com 
李子杰 江南大学生物工程学院  
高晓冬 江南大学生物工程学院 xdgao@jiangnan.edu.cn 
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中文摘要:
      【目的】D-阿洛酮糖-3-差向异构酶(D-psicose-3-epimerase,DPE)可以催化D-果糖和D-阿洛酮糖之间的可逆反应,但D-阿洛酮糖的产率较低。L-鼠李树胶糖激酶(L-rhamnulose kinase,RhaB)具有磷酸化C3-R构型酮糖的活性,将其与DPE偶联,可以打破DPE的可逆平衡,从而提高D-阿洛酮糖的产率。在反应体系中引入多聚磷酸盐激酶(polyphosphate kinase,PPK),可实现ATP的再生,降低反应的成本。【方法】本实验在大肠杆菌Rosetta(DE3)中分别过表达DPE、RhaB和PPK,优化其反应条件,计算产物得率。【结果】结果表明,RhaB的分子量约为53 kDa,最适pH和温度为8.0和35℃。将DPE和RhaB偶联,最适条件如下:pH 8.5,温度35℃,最适金属离子Mg2+,DPE和RhaB酶量比1:2,产物得率为70%。将DPE、RhaB和PPK偶联,ATP浓度降为初始浓度的1/5,D-阿洛酮糖得率为50%。【结论】本研究为工业化生产D-阿洛酮糖提供理论依据。
英文摘要:
      D-psicose can be produced from D-fructose catalyzed by D-psicose-3-epimerase (DPE). However, the reaction catalyzed by DPE is reversible, which leads to a low conversion rate of D-psicose. L-rhamnulose kinase (RhaB) catalyzes the phosphorylation of C3-R ketoses in an irreversible way. In the cascade reaction combined with DPE, RhaB can shift the equilibrium by phosphorylation of D-psicose. Thus, the production of D-psicose with increased rate can be expected. In order to reduce the synthetic costs, polyphosphate kinase (PPK) was overexpressed in the cascade reaction to achieve the regeneration of ATP. DPE, RhaB and PPK were overexpressed in E. coli Rosetta (DE3) respectively and the optimum conditions for the enzymatic activity were investigated. The analysis of SDS-PAGE revealed the molecular weight of RhaB was 53 kDa, and its optimum pH and temperature were determined as 8.0 and 35℃. In the cascade reaction combined with DPE, the optimum conditions were also tested and determined as follows: a) the values of pH and temperature were 8.5 and 35℃; b) Mg2+ was the most optimum metal ion for the reaction; c) the optimum ratio for DPE and RhaB was 1:2. As a consequence, the yield of D-psicose could reach 70%. In the cascade reaction combined with PPK, the ATP concentration was reduced to 1/5 of the original amount and the yield of D-psicose was 50%. The results could provide the theoretical basis for the industrial production of D-psicose.
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