Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity
Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity
复制标题
DOI：
10.1007/s00253-011-3201-y
复制
发表时间：
2011-07-01
影响因子：
5
通讯作者：
Kimura, Atsuo
中科院分区：
文献类型：
--
作者：
Kim, Young-Min;Shimizu, Ryoko;Kimura, Atsuo
存在糖苷水解酶家族（GH）66的多种形式的天然和重组内切葡聚糖酶（Dexs）。变形链球菌GH 66 Dex基因在大肠杆菌中表达.将重组全长（95.4kDa）SmDex蛋白消化以形成89.8kDa同种型（SmDex 90）。纯化的SmDex 90在长期储存期间被蛋白水解降解为超过7种多肽（23-70 kDa）。该蛋白酶不敏感性蛋白为SmDex的生化分析和利用提供了理想的蛋白质。预计GH 66 Dex从N-至C-末端包含4个区域：N-末端可变区（N-VR）、保守区（CR）、葡聚糖结合位点（GBS）和C-末端可变区（C-VR）。通过删除N-VR、GBS和/或C-VR产生五个截短的SmDex。两种缺乏C-VR（TM-NCG Delta）或N-VR/C-VR（TM-Delta CG Delta）的截短突变体酶具有催化活性，从而表明N-VR和C-VR对于催化活性不是必需的。在长期储存期间，TM-Delta CG Delta不接受任何进一步的蛋白酶降解。TM-NCG Delta和TM-Delta CG Delta增强底物水解，表明N-VR和C-VR诱导底物与活性位点的受阻结合。
Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.