College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, Hunan, China.
School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore, 599489, Singapore.
Mol Biotechnol. 2019 Jun;61(6):432-441. doi: 10.1007/s12033-019-00174-6.
D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.
D-阿洛酮糖是一种罕见的单糖，在自然界中存在的量极微，且由于转化率低和下游分离复杂，很难通过化学或酶合成途径大规模制备。最近，利用 D-阿洛酮糖差向异构酶和 L-鼠李糖激酶，建立了一种无需繁琐的异构分离步骤即可从 D-果糖高转化率制备 D-阿洛酮糖的方法。然而，该方法需要昂贵的 ATP 来促进反应。在本研究中，通过与多聚磷酸激酶偶联开发了一种 ATP 再生系统。在优化的酶促反应中，在 50°C 和 pH7.5 条件下，当 2 mM ATP、5 mM 多聚磷酸、20 mM D-果糖和 20 mM Mg 存在时，99% D-果糖的转化率达到 99%。与没有 ATP 再生系统相比，ATP 的使用量可减少至理论用量的 10%。还研究了补料分批模式以最小化多聚磷酸的抑制作用。本报告的生物合成系统为降低 ATP 成本将 D-果糖转化为 D-阿洛酮糖提供了一个有潜力和有前途的平台。
