State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Department of Biotechnology and Enzyme Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 25, D-70599, Stuttgart, Germany.
Appl Biochem Biotechnol. 2019 Apr;187(4):1255-1271. doi: 10.1007/s12010-018-2877-1. Epub 2018 Sep 14.
Trehalose is a non-reducing disaccharide with beneficial physiological properties and commercial potential. Trehalose synthase (EC 5.4.99.16) catalyzes the reversible conversion between maltose and trehalose. A recombinant trehalose synthase from Arthrobacter chlorophenolicus SK 33.001 (ACTS) was cloned, expressed, and characterized. The recombinant enzyme encoded a protein of 598 amino acids with a molecular mass of 66 kDa. Gel filtration showed that ACTS is a tetramer in sodium phosphate buffer. The enzyme was metal ion independent and exhibited maximal activity in sodium phosphate buffer (pH 7.5) at 30 °C. The kinetic investigations resulted in a K value of 120.5 ± 4.5 mM for maltose and a K value of 343.1 ± 13.8 mM for trehalose. The catalytic efficiency (V/K) for maltose and trehalose were 0.2 and 0.15 U mg mM, respectively. In addition, a cooperative substrate binding was found displayed by the determined Hill coefficients (n) of 2.8 for maltose and 2.1 for trehalose as a substrate, respectively. The final trehalose yield of various maltose concentrations (50-1000 mM) was constant between 58 and 59%, implying that substrate concentration had no inhibitory influence on ACTS activity.
海藻糖是一种具有有益生理特性和商业潜力的非还原二糖。海藻糖合酶（EC 5.4.99.16）催化麦芽糖和海藻糖之间的可逆转化。从节杆菌属（Arthrobacter）绿脓杆菌 SK 33.001（ACTS）克隆、表达和表征了一种重组海藻糖合酶。该重组酶编码一个 598 个氨基酸的蛋白质，分子量为 66 kDa。凝胶过滤显示 ACTS 在磷酸钠缓冲液中为四聚体。该酶是金属离子非依赖性的，在 30°C 时在磷酸钠缓冲液（pH 7.5）中表现出最大活性。动力学研究得出，麦芽糖的 K 值为 120.5±4.5 mM，海藻糖的 K 值为 343.1±13.8 mM。麦芽糖和海藻糖的催化效率（V/K）分别为 0.2 和 0.15 U mg mM。此外，作为底物，麦芽糖和海藻糖的测定 Hill 系数（n）分别为 2.8 和 2.1，表明存在协同底物结合。在各种麦芽糖浓度（50-1000 mM）下，最终海藻糖产量在 58%至 59%之间保持恒定，这表明底物浓度对 ACTS 活性没有抑制影响。
