State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
Appl Microbiol Biotechnol. 2014 Jan;98(2):717-25. doi: 10.1007/s00253-013-4924-8. Epub 2013 May 4.
D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using D-psicose as the substrate, the apparent K(m), k(cat), and catalytic efficiency (k(cat)/K(m)) were 27.4 mM, 49 s⁻¹, and 1.78 s⁻¹ mM⁻¹, respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production.
D-塔格糖 3-差向异构酶家族的酶可以有效地催化游离酮糖的差向异构化，这可用于从 D-果糖生产 D-阿洛酮糖。在以前的研究中，这些酶的所有最适 pH 值都被发现为碱性。在本研究中，从 Clostridium bolteae（ATCC BAA-613）中鉴定并表征了一种具有中性 pH 最适值的 D-阿洛酮糖 3-差向异构酶（DPEase）。编码重组 DPEase 的基因被克隆并在大肠杆菌中表达。为了表征催化特性，使用镍亲和层析将重组 DPEase 纯化至电泳均一性。乙二胺四乙酸被证明完全抑制酶活性；因此，该酶被鉴定为一种金属蛋白酶，在存在 Co²⁺的情况下表现出最高的活性。尽管 DPEase 在 pH 值为 6.5 至 7.5 之间表现出最高的活性，但它在 pH 值为 7.0 时表现出最佳的活性。重组 DPEase 的最适温度为 55°C，半衰期为 156 分钟，在 55°C 下。使用 D-阿洛酮糖作为底物，表观 K(m)、k(cat)和催化效率（k(cat)/K(m)）分别为 27.4 mM、49 s⁻¹和 1.78 s⁻¹ mM⁻¹。在最佳条件下，D-果糖与 D-阿洛酮糖的平衡比为 69:31。为了高产 D-阿洛酮糖，在 pH 值为 6.5 和 55°C 下，可生产 216 g/L 的 D-阿洛酮糖，转化率为 28.8%。重组 DPEase 表现出弱酸性稳定性和热稳定性，对底物 D-果糖具有高亲和力和周转率，表明该酶是工业生产 D-阿洛酮糖的潜在酶制剂。
