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

作　　者：王利飞 谭子瑊 谢希贤[1] 朱蕾蕾 WANG Lifei;TAN Zijian;XIE Xixian;ZHU Leilei(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Key Laboratory of Engineering Biology for Low-carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,Jiangsu,China)

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]中国科学院天津工业生物技术研究所低碳合成工程生物学重点实验室,天津300308 [3]南京中医药大学药学院,江苏南京210023

出　　处：《微生物学报》2023年第11期4197-4207,共11页Acta Microbiologica Sinica

基　　金：天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-003-23)。

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57.9kDa,比活力为0.9U/mg,最适反应温度和pH值分别为70°C和9.0,最适金属离子为Ni2+,最适金属离子浓度为2mmol/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。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°C 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°C,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.

关 键 词：D-塔格糖 塔格糖-4-差向异构酶 果糖异构化 D-果糖 

分 类 号：Q814[生物学—生物工程]