Liu Zhanzhi, Li Ying, Wu Jing, Chen Sheng
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
Foods. 2020 Dec 6;9(12):1809. doi: 10.3390/foods9121809.
d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene of -acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from was amplified, and the recombinant AGEase was characterized. The optimal temperature and pH of AGEase were 60 °C and 7.5, respectively. The , , and / of AGEase for d-mannose were 49.2 ± 8.5 mM, 476.3 ± 4.0 s, and 9.7 ± 0.5 s·mM, respectively. The recombinant AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the AGEase belongs to the d-mannose isomerase of the YihS enzyme. The AGEase catalyzed the synthesis of d-mannose with d-fructose as a substrate, and the conversion rate was as high as 39.3% with the d-mannose yield of 78.6 g·L under optimal reaction conditions of 200 g·Ld-fructose and 2.5 U·mL AGEase. This novel AGEase has potential applications in the industrial production of d-mannose.
D-甘露糖在食品、制药和饲料行业已展现出优异的生理特性。因此，由于其相较于化学合成的优势，酶法生产D-甘露糖受到了越来越多的关注。扩增了源自[具体来源未提及]的β-乙酰-D-葡萄糖胺2-表异构酶家族表异构酶/异构酶（AGEase）基因，并对重组AGEase进行了表征。AGEase的最适温度和pH分别为60℃和7.5。AGEase对D-甘露糖的Km、Vmax和kcat/Km分别为49.2±8.5 mM、476.3±4.0 s和9.7±0.5 s·mM。通过分析其底物特异性和三维（3D）结构的活性中心，将重组AGEase归类为AGE酶家族中的YihS酶亚家族。对不同底物动力学的进一步研究表明，AGEase属于YihS酶的D-甘露糖异构酶。AGEase以D-果糖为底物催化合成D-甘露糖，在200 g·L D-果糖和2.5 U·mL AGEase的最佳反应条件下，转化率高达39.3%，D-甘露糖产量为78.6 g·L。这种新型AGEase在D-甘露糖的工业生产中具有潜在应用。
