Mislovicová D, Masárová J, Hostinová E, Gasperík J, Gemeiner P
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia.
Int J Biol Macromol. 2006 Nov 15;39(4-5):286-90. doi: 10.1016/j.ijbiomac.2006.04.005. Epub 2006 Apr 28.
Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.
制备了各种类型的糖化酶以调节它们与伴刀豆球蛋白A的生物特异性相互作用。糖化酶Glm是从天然酵母菌株扣囊复膜孢酵母IFO 0111中分离得到的。表达并分离了来自扣囊复膜孢酵母HUT 7212的两种糖基化重组糖化酶Glu，分别来自酿酒酵母菌株，还有一种非糖基化的来自大肠杆菌。研究了这些制剂对伴刀豆球蛋白A的生物特异性亲和力，并与市售的黑曲霉真菌糖化酶GA进行了比较。所有糖基化酶都表现出对伴刀豆球蛋白A的亲和力，其特征在于它们的沉淀过程以及平衡解离常数在1.43至4.17×10(-6)M范围内（通过SPR方法测定）。结果表明伴刀豆球蛋白A与各个糖化酶的相互作用存在一些差异。对伴刀豆球蛋白A亲和力最高的是GA。糖含量较高的重组糖化酶Glu由两个具有不同亲和力的结合位点组成。亲和力最低的糖化酶（Glm和糖含量较低的Glu）在沉淀实验中也表现出与伴刀豆球蛋白A的非特异性相互作用。通过α-D-甘露吡喃糖苷的抑制实验确定了各个糖化酶之间的最小差异。
