Sugimoto Manabu, Ohta Takeshi, Kawai Fusako
Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Okayama 710-0046, Kurashiki, Japan.
Biochim Biophys Acta. 2003 Jan 31;1645(1):1-5. doi: 10.1016/s1570-9639(02)00525-3.
The chimeric alpha-glucosidases of Mucor javanicus and Aspergillus oryzae, which has high activity toward not only maltooligosaccharides but also soluble starch and has high activity toward maltooligosaccharides but faint activity toward soluble starch, respectively, were constructed by shuffling the C-terminal regions where low homology is observed between the two enzymes. The chimera genes were expressed in Pichia pastoris to produce and secrete the enzymes that have predicted molecular masses in the culture medium. The two chimeric M. javanicus alpha-glucosidases, of which the N- and C-terminal regions are substituted for those of A. oryzae, respectively, decreased in soluble starch-hydrolyzing activity, however, increased in maltose-hydrolyzing activity by 2.1 and 4.9 times higher than that of the native form of M. javanicus alpha-glucosidase, respectively. The chimeric enzymes changed on the V(max) values for maltose significantly, whereas the K(m) values were similar to that of the native enzyme.
通过改组米根霉和米曲霉α-葡萄糖苷酶的C端区域（这两个酶的该区域间同源性较低），构建了嵌合α-葡萄糖苷酶。改组后的米根霉嵌合α-葡萄糖苷酶不仅对麦芽寡糖具有高活性，对可溶性淀粉也具有高活性；改组后的米曲霉嵌合α-葡萄糖苷酶对麦芽寡糖具有高活性，但对可溶性淀粉活性较弱。将嵌合基因在毕赤酵母中表达，以便在培养基中产生和分泌具有预测分子量的酶。两种N端和C端区域分别被米曲霉相应区域取代的米根霉嵌合α-葡萄糖苷酶，其可溶性淀粉水解活性降低，但麦芽糖水解活性分别比天然米根霉α-葡萄糖苷酶提高了2.1倍和4.9倍。嵌合酶的麦芽糖V(max)值发生了显著变化，而K(m)值与天然酶相似。
