学术
耻垢分枝杆菌海藻糖合成酶基因TreS的克隆及其在大肠杆菌中的表达
李镭[1,2] 丁宏标[2] 余晓斌[1] 乔宇[2]
[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]中国农业科学院饲料研究所,北京100081
摘 要:
耻垢分枝杆菌（Mycobacterium smegmatis）在环境胁迫下具有合成海藻糖的能力。实验采用PCR的方法，克隆了来源于耻垢分枝杆菌的一段核苷酸序列，与已报道的海藻糖合成酶有60％以上同源性，推测该基因的编码产物具有海藻糖合成酶的活性，为进行其功能验证而在大肠杆菌中进行了表达。目的蛋白以可溶性蛋白为主，约占细胞可溶性总蛋白48％，为目前相关报道的最高值。经活性测定，表达的重组酶无需复性，能够催化麦芽糖生成海藻糖，在20℃反应20h对麦芽糖的转化率可达61％，具有较高的应用价值。[著者文摘]
文章出处:
《食品与生物技术学报》-2007年26卷6期 -69-73页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1673-1689（2007）06-0069-05
Cloning and Expression of TreS Gene from Mycobacterium smegmatis in Escherichia coli
LI Lei, DING Hong-biao, YU Xiao-bin, QIA0 Yu（1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, WuXi 214122, China; 2. Feed Research Institute, China Academy of Agricultural Sciences, Beijing 100081, China）
Abstract:
Trehalose synthase catalyzes the reversible interconversion of maltose and trehalose （1- O-α-D-glucopyranosyl-α-D-glucopyranoside ）. An trehalose synthase gene, Tres from Mycobacterium smegmatis,has been cloned by PCR. The sequence encoding TreS was inserted into vector pET30a and introduced into the host Escherichia coli BL21（DE3）pLysS. A high yield of the active recombinant TreS,48% of total protein, was obtained by IPTG induction. A novel protein band of 68kDa was detected by SDS-PAGE analysis. It was found that the optimum reaction time for recombinant TreS about 20h. Higher conversion yield was observed between 4℃～30℃,and, more than 61% conversion yield was obtained at 20℃. The addition of lmM divalent metal ions such as Ba^2＋ . Ca^2＋ , Co^2＋ . Mg^2＋ , Mn^2＋ , did not further increase the enzyme activity,However Cu^2＋ strongly inhibited the enzyme activity.[著者文摘]
Key words:
Mycobacterium smegmatis; trehalose synthase; cloning; expression
基金资助:
国家863计划项目（2005AA246010）;国际科技合作重点项目（2005DFA31070）.
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