D-塔格糖是一种稀有的具有特殊保健功能的天然食品甜味剂。目前,以D-半乳糖为底物,基于异构化途径的D-塔格糖生物合成受到基本的热力学平衡限制,底物转化率较低;而基于氧化还原途径,可通过热力学耦合辅因子避免不必要的逆反应,具有更高的理论转化率;但现有氧化还原途径中关键酶醛糖还原酶底物谱广,对D-半乳糖底物特异性低,是制约此合成工艺发展的瓶颈。该研究以Scheffersomyces stipitis CBS 6054来源木糖还原酶(xylose reductase,XR)SsXR为模板,基于氨基酸同源性分析,从美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)中筛选出11个同源序列,并分别对其进行同源建模和分子对接,以D-半乳糖与蛋白分子的结合自由能、作用位点处氨基酸距离为主,结合底物在蛋白口袋中的深度及契合程度等要素筛选到对D-半乳糖特异性最佳的Spathaspora gorwiae来源SgXR。将SsXR和SgXR分别引入大肠杆菌中异源表达并进行酶学性质研究。结果显示,二者均为烟酰胺腺嘌呤二核苷酸磷酸氢(nicotinamide adenine dinucleotide phosphate hydrogen,NADPH)偏好,SgXR对D-半乳糖的底物亲和力是SsXR的4倍,其Km值与kcat分别为SsXR的1/4与1.11倍;进一步引入来源Rhizobium legumenosarum的半乳糖醇脱氢酶RlGDH,以10 g/L D-半乳糖为底物,发酵48 h,含有SgXR的重组菌D-塔格糖产量达到5.47 g/L,是SsXR的1.23倍,且对D-半乳糖转化率为54.7%,高于大多数基于异构化途径的报道。该研究为基于氧化还原途径的D-塔格糖高效生物合成工艺开发奠定了基础。
D-tagatose is a rare natural food sweetener with special health care functions. Currently, the isomerization pathway for D-tagatose production using D-galactose as substrate is limited by the basic thermodynamic equilibrium, which often leads to low substrate conversion rate. However, D-tagatose biosynthesis based on redox pathway can avoid unnecessary reverse reactions by thermodynamically coupling cofactors and has a higher theoretical conversion rate. While, the existing aldose reductase, the key enzyme of the redox pathway, has a wide substrate spectrum and is not specific for substrate D-galactose, which is the bottleneck that restricts the development of this bioprocess. In this study, the xylose reductase from Scheffersomyces stipitis CBS 6054 (SsXR) was used as the template, and 11 homologous sequences were screened from National Center for Biotechnology Information (NCBI) based on amino acid homology analysis, and homology modeling and molecular docking were performed. Based on the binding free energy and the distance of action sites between D-galactose and protein molecule, the depth and matched-degree of substrate in protein pockets, the XR from Spathaspora gorwiae (SgXR) with highest substrate specificity was selected. SsXR and SgXR were expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the two purified enzymes were studied. The substrate specificity of SgXR for D-galactose was 4-times that of SsXR. The Km value and kcat of SgXR for D-galactose were 1/4 and 1.11 times that of SsXR, respectively. Furthermore, by introducing galactitol dehydrogenase RlGDH from Rhizobium legumenosarum, the recombinant strain containing SgXR produced 5.47 g/L D-tagatose from 10 g/L D-galactose within 48 h, which was 1.23 times higher than that of SsXR. The D-tagatose yield from D-galactose was 54.7%, which was higher than most reports based on isomerization pathways. These results laid a foundation for the development of efficient D-tagatose biosynthesis process based on redox pathway.
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