Li Runjing, Zhang Tao, Jiang Bo, Mu Wanmeng, Miao Ming
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, People's Republic of China.
Biotechnol Appl Biochem. 2015 Nov-Dec;62(6):815-22. doi: 10.1002/bab.1334. Epub 2015 Mar 27.
An intracellular levansucrase from Bacillus methylotrophicus SK 21.002 was isolated, purified, and characterized. The final specific levansucrase activity was 135.40 U/mg protein with an 11.78-fold enrichment and a 9.28% recovery rate. The molecular weight of the enzyme was approximately 60,000 Da as evaluated by gel filtration and SDS-PAGE. Both the maximum transfructosylation and hydrolytic activities were observed at pH 6.5. The enzyme exhibited optimum transfructosylation activity at 40 °C, whereas the optimum temperature of hydrolytic activity was 45 °C. Cu(2+), Fe(2+), Zn(2+), and Ni(2+) inhibited both the transfructosylation and hydrolytic activities up to 100%, whereas Mn(2+) inhibited only hydrolytic activity. Ca(2+) and Mg(2+) stimulated both transfructosylation and hydrolytic activities. The chemical modifiers (n-bromosuccinimide and phenylmethanesulfonyl fluoride) strongly inhibited hydrolytic and transfructosylation activity of the levansucrase. The Km and Vmax values of the purified levansucrase were 117.2 mM and 33.23 μmol/mg·Min, respectively. When the fructose concentration was below 0.2 M, higher fructose concentrations promoted the transfructosylation and inhibited the hydrolytic activity.
从甲基营养芽孢杆菌SK 21.002中分离、纯化并表征了一种细胞内蔗糖酶。最终的比蔗糖酶活性为135.40 U/mg蛋白，富集倍数为11.78倍，回收率为9.28%。通过凝胶过滤和SDS-PAGE评估，该酶的分子量约为60,000 Da。在pH 6.5时观察到最大转果糖基化和水解活性。该酶在40°C时表现出最佳转果糖基化活性，而水解活性的最佳温度为45°C。Cu(2+)、Fe(2+)、Zn(2+)和Ni(2+)对转果糖基化和水解活性的抑制率高达100%，而Mn(2+)仅抑制水解活性。Ca(2+)和Mg(2+)刺激转果糖基化和水解活性。化学修饰剂（N-溴代琥珀酰亚胺和苯甲基磺酰氟）强烈抑制蔗糖酶的水解和转果糖基化活性。纯化的蔗糖酶的Km和Vmax值分别为117.2 mM和33.23 μmol/mg·Min。当果糖浓度低于0.2 M时，较高的果糖浓度促进转果糖基化并抑制水解活性。
