阅读说明：本技术 一种利用全细胞转化合成d-阿洛酮糖的方法 (Method for synthesizing D-psicose by whole-cell transformation ) 是由 朱理平 高晓冬 李子杰 温俊婷 冯林雪 陈洲 陈科材 于 2020-05-08 设计创作，主要内容包括：本发明涉及生物技术领域,尤其涉及一种利用全细胞转化合成D-阿洛酮糖的方法,所述方法中整个反应体系包括底物D-果糖,重组大肠杆菌经过诱导后的湿菌体、ATP以及金属离子,所述反应体系经过催化反应后再加热,经过降温后加入酸性磷酸酶经过脱磷酸反应得到D-阿洛酮糖。利用该方法可提高D-阿洛酮糖的产率。(The invention relates to the technical field of biology, in particular to a method for synthesizing D-psicose by whole cell transformation. The method can improve the yield of D-psicose.)

1. A method for synthesizing D-psicose by whole-cell transformation, which is characterized by comprising the following steps: the whole reaction system comprises a substrate D-fructose, wet thalli, ATP and metal ions after induction of recombinant escherichia coli, the reaction system is heated after catalytic reaction, acid phosphatase is added after temperature reduction, and D-psicose is obtained through dephosphorylation reaction.

2. The method for synthesizing D-psicose using whole-cell transformation according to claim 1, wherein: the pH value of the whole reaction system is 7-10, the reaction temperature is 20-60 ℃, the reaction time is 2-6h, and the metal ions are Mg²⁺、Mn²⁺、Co²⁺Or Ca²⁺(ii) a The reheating temperature after the catalytic reaction is controlled to be 80-100 ℃, and the temperature after the temperature reduction is controlled to be 20-37 DEG C。

3. The method for synthesizing D-psicose using whole-cell transformation according to claim 2, wherein: the pH value of the whole reaction system is 8.5 during the catalytic reaction, the reaction temperature is 37 ℃, and the metal ion is Mg²⁺。

4. The method for synthesizing D-psicose using whole-cell transformation according to claim 2, wherein: the recombinant escherichia coli is a recombinant escherichia coli which can simultaneously express L-rhamnose kinase and D-psicose-3-epimerase.

5. The method for synthesizing D-psicose using whole-cell transformation according to claim 4, wherein: the construction method of the recombinant escherichia coli comprises the following steps: connecting the L-rhamnogum kinase gene rhaB to pET28a plasmid to obtain recombinant plasmid pET28 a-rhaB; connecting the D-psicose-3-epimerase gene dpe to pCDFDuet-1 plasmid to obtain recombinant plasmid pCDFDuet-dpe; then pET28a-rhaB and pCDFDuet-dpe are jointly transformed into Escherichia coli Roseta (DE3) to obtain recombinant Escherichia coli; or the single plasmid pCDFDuet-1 is used for simultaneously inserting rhaB and dpe genes to obtain a recombinant plasmid pCDFDuet-rhaB-dpe, and then the recombinant plasmid pCDFDuet-rhaB-dpe is transformed into escherichia coli Roseta (DE3) to obtain the recombinant escherichia coli.

6. The method for synthesizing D-psicose using whole-cell transformation according to claim 5, wherein: the recombinant escherichia coli is cultured in an LB culture medium at 37 ℃, when OD600 is 0.6-1.0, 0.1-1.0mM IPTG is added in final concentration, induction is carried out for 5-20h at 16-37 ℃, and wet thalli are collected.

7. The method for synthesizing D-psicose using whole-cell transformation according to claim 2, wherein: the recombinant escherichia coli is a recombinant escherichia coli which can simultaneously express L-rhamnose gum kinase, D-psicose-3-epimerase and polyphosphate kinase; when the recombinant escherichia coli is adopted, the whole reaction system also comprises sodium polyphosphate.

8. The method for synthesizing D-psicose using whole-cell transformation according to claim 7, wherein: the construction method of the recombinant escherichia coli comprises the following steps: connecting the L-rhamnogum kinase gene rhaB and polyphosphate kinase gene ppk1 to pCDFDuet-1 plasmid to obtain recombinant plasmid pCDFDuet-rhaB-ppk; connecting the D-psicose-3-epimerase gene dpe to pET28a plasmid to obtain recombinant plasmid pET28 a-dpe; then pET28a-dpe and pCDFDuet-rhaB-ppk recombinant plasmid were co-transformed into E.coli Roseta (DE3) to obtain recombinant E.coli.

9. The method for synthesizing D-psicose using whole-cell transformation according to claim 8, wherein: culturing the recombinant escherichia coli in an LB culture medium at 37 ℃ until OD600 is 0.6-1.0, adding 0.1-1.0mM IPTG (Methylobacillus thuringiensis) with final concentration, inducing at 16-37 ℃ for 5-20h, and collecting wet thalli.