Guo Yan, Feng Tingting, Wang Zhiqi, Li Hongwei, Wei Xin, Chen Jing, Niu Debao, Liu Jidong
College of Light Industry and Food Engineering, Guangxi University, 100 Daxue Road, Nanning 530004, Guangxi, China.
Guangxi South Subtropical Agricultural Sciences Research Institute, Longzhou, Guangxi 532415, China.
J Agric Food Chem. 2022 Dec 14;70(49):15539-15547. doi: 10.1021/acs.jafc.2c06920. Epub 2022 Dec 2.
d-Allulose is a desirable sucrose substitute with potential applications in food and health care. d-Allulose can be synthesized using d-glucose as a substrate through coupling glucose isomerase with d-allulose 3-epimerase (DAEase); however, the product yield is typically less than 20% at reaction equilibrium and thus limits its use in industrial applications. Here, a 3R-ketose phosphorylation pathway coupled with an adenosine triphosphate (ATP) regeneration system was developed for the efficient synthesis of d-allulose in using d-glucose as a substrate. The l-rhamnulose kinase (RhaB) was used to break the inherent reaction equilibrium due to its substrate specificity, resulting in increases in d-allulose titer by 69.9% to 4.96 ± 0.49 g/L. By optimizing the whole cell transformation conditions and designing an ATP regeneration module, d-allulose production reached 17.62 ± 0.77 g/L from 30 g/L d-glucose with a final yield of 0.73 g/g without the addition of exogenous ATP. To evaluate the potential industrial application of this multienzyme cascade system, d-allulose was produced from cane molasses (124.16 ± 2.69 g/L glucose equivalent) with a final d-allulose titer of 62.60 ± 3.76 g/L. The present study provides a practical enzymatic approach for the economical synthesis of d-allulose.
D-阿洛酮糖是一种理想的蔗糖替代品，在食品和医疗保健领域具有潜在应用价值。D-阿洛酮糖可以以D-葡萄糖为底物，通过将葡萄糖异构酶与D-阿洛酮糖3-差向异构酶（DAEase）偶联来合成；然而，在反应平衡时产物收率通常低于20%，因此限制了其在工业应用中的使用。在此，开发了一种与三磷酸腺苷（ATP）再生系统偶联的3R-酮糖磷酸化途径，用于以D-葡萄糖为底物高效合成D-阿洛酮糖。利用L-鼠李糖激酶（RhaB）因其底物特异性打破固有的反应平衡，使D-阿洛酮糖滴度提高了69.9%，达到4.96±0.49 g/L。通过优化全细胞转化条件并设计ATP再生模块，在不添加外源ATP的情况下，以30 g/L D-葡萄糖为原料，D-阿洛酮糖产量达到17.62±0.77 g/L，最终产率为0.73 g/g。为了评估这种多酶级联系统的潜在工业应用，从甘蔗糖蜜（124.16±2.69 g/L葡萄糖当量）中生产D-阿洛酮糖，最终D-阿洛酮糖滴度为62.60±3.76 g/L。本研究为经济合成D-阿洛酮糖提供了一种实用的酶法途径。
