塔格糖-4-异构酶的新酶挖掘及酶学性质研究

Discovery and characterization of a novel tagatose-4-epimerase

摘要:

d-塔格糖是一种己酮糖，在自然界中天然存在但含量极少，具有降血糖、抗龋齿、改善肠道菌群、促进血液循环和抗衰等作用，可广泛应用于食品、医药和化妆品行业。目前酶催化半乳糖生成塔格糖仅需一步反应，简单易行，但半乳糖较高的成本限制了这一方法的工业应用。果糖是塔格糖的同分异构体，价格低廉，来源稳定，可通过4位差向异构化一步获得塔格糖，是产塔格糖的理想底物。【目的】因此挖掘新的塔格糖-4-差向异构酶对塔格糖的工业生产具有重要意义。【方法】本文通过基因挖掘的手段发现了热袍菌(Thermotogae bacterium)来源的未知功能蛋白(命名为HCZ0)具有塔格糖-4-差向异构酶活性，进而完成了HCZ0的异源表达、纯化及酶学性质表征。【结果】确定HCZ0表观分子量约为57.9 kDa，比活力为0.9 U/mg，最适反应温度和pH值分别为70 ℃和9.0，最适金属离子为Ni²⁺，最适金属离子浓度为2 mmol/L，60、70和80 ℃半衰期分别为180、67和9 h。最适条件下，HCZ0催化200 g/L果糖在2 h内可产生28 g/L塔格糖。【结论】本研究中，HCZ0是碱性高温酶，且具有较好的热稳定性，这些特征在以后的理论研究及工业生产中具有一定的科学价值。

Abstract:

d-tagatose is a natural and rare ketohexose with blood glucose-lowering, anti-caries, gut microbiome-regulating, blood circulation-promoting, and anti-aging activities, and thus it is widely applied in food, pharmaceutical, and cosmetic industries. Though the enzymatic conversion of galactose into tagatose is a simple process, the high cost of galactose limits its industrial application. Fructose, an isomer of tagatose, is an economical alternative with stable supply. Through 4-epimerization, tagatose can be produced from fructose, which is an ideal substrate for tagatose production. [Objective] To discover new tagatose-4-epimerases essential for the efficient industrial production of tagatose. [Methods] Gene mining was performed to identify an unknown protein HCZ0 with 4-epimerization activity from Thermotogae bacterium. HCZ0 was heterologously expressed, purified, and characterized. [Results] HCZ0 had a molecular weight of 57.9 kDa and the specific activity of 0.9 U/mg. The enzyme showed the highest activity at 70 ℃ and pH 9.0, and it activity was significantly improved by 2 mmol/L Ni²⁺. HCZ0 exhibited the half-life of 180, 67, and 9 h at 60, 70, and 80 ℃, respectively. Under optimal conditions, the HCZ0 was able to catalyze 200 g/L fructose to produce 28 g/L tagatose within 2 h. [Conclusion] HCZ0 is an alkaline high-temperature enzyme with good thermal stability, which can provide inspiration for future theoretical research and industrial production.