Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E-coli and characterization of the recombinant enzyme
Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E-coli and characterization of the recombinant enzyme
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DOI：
10.1023/a:1025035320983
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发表时间：
2003-08-01
影响因子：
2.7
通讯作者：
Lee, JH
中科院分区：
文献类型：
--
作者：
Kim, MJ;Kwon, T;Lee, JH
从经EcoRI酶切的长双歧杆菌SJ32基因组文库中分离出一段DNA片段，该片段在含有棉子糖的M9培养基和含有氨苄西林、IPTG和5-溴-4-氯-3-吲哚- α - d -半乳糖苷的LB培养基上均能补充大肠杆菌的生长。在克隆的DNA片段中，通过缺失分析和序列分析，鉴定了一个编码蔗糖磷酸化酶（splP）的基因和一个部分克隆的假定蔗糖调控基因（splR）。在大肠杆菌中合成了一个56 kDa的蛋白，并通过deae -离子交换层析进行了部分纯化。部分纯化的酶不能与糖醛酸糖、糖醛酸糖和棉子糖反应，但能与蔗糖反应。它除了具有水解活性外，还具有转糖基化活性。
A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl-alpha-D-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.