Funane Kazumi, Ishii Tadashi, Ono Hiroshi, Kobayashi Mikihiko
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
FEBS Lett. 2005 Aug 29;579(21):4739-45. doi: 10.1016/j.febslet.2005.07.050.
Dextransucrase S (DSRS) is the only active glucansucrase that has been found in Leuconostoc mesenteroides NRRL B-512F strain. Native DSRS produces mainly 6-linked glucopyranosyl residue (Glcp), while Escherichia coli recombinant DSRS was observed to produce a glucan consisting of 70% 6-linked Glcp and 15% 3,6-Glcp. Lys residues were introduced at the N-terminal end of the core domain by site-directed mutagenesis. In glucans produced by the one-point mutants T350K and S455K, the amount of 6-linked Glcp was increased to about 85% of the total glucan produced, more similar in structure to native B-512F dextran. The double mutant T350K/S455K produced adhesive, water-insoluble glucan with 77% 6-linked Glcp, 8% 3,6-linked Glcp and 4% 2,6-linked Glcp. The T350K/S455K mutant exhibited a 10-fold increase in glucosyltransferase activity over those of the parental DSRS-His(6) and its T350K and S455K mutants. This is the first report demonstrating a change in the properties of a dextransucrase or a related glucosyltransferase through simple site-directed mutagenesis to create 2,6-linked Glcp.
葡聚糖蔗糖酶S（DSRS）是在肠膜明串珠菌NRRL B - 512F菌株中发现的唯一一种活性葡聚糖蔗糖酶。天然DSRS主要产生6 - 连接的葡萄糖吡喃糖基残基（Glcp），而观察到大肠杆菌重组DSRS产生的葡聚糖由70%的6 - 连接Glcp和15%的3,6 - Glcp组成。通过定点诱变在核心结构域的N末端引入赖氨酸残基。在单点突变体T350K和S455K产生的葡聚糖中，6 - 连接Glcp的量增加到所产生的总葡聚糖的约85%，其结构更类似于天然B - 512F葡聚糖。双突变体T350K/S455K产生了具有粘性的、水不溶性葡聚糖，其中含有77%的6 - 连接Glcp、8%的3,6 - 连接Glcp和4%的2,6 - 连接Glcp。与亲本DSRS - His(6)及其T350K和S455K突变体相比，T350K/S455K突变体的葡糖基转移酶活性提高了10倍。这是首次通过简单的定点诱变来产生2,6 - 连接Glcp从而证明葡聚糖蔗糖酶或相关葡糖基转移酶性质发生变化的报道。
