Yang Sung Jae, Park Na Hee, Lee Tae Ho, Cha Jaeho
Department of Microbiology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Kumjung-ku, Busan, Korea.
Biotechnol Appl Biochem. 2002 Jun;35(3):199-203.
A 1.6 kb DNA fragment including the lftM gene, encoding a levan fructotransferase (LFTase) of Microbacterium sp. AL-210, was subcloned into a high-expression vector, pET-29b, and the recombinant enzyme was overexpressed in Escherichia coli. Most of the LFTase activity was detected in the cytoplasmic fraction after induction with isopropyl beta-d-thiogalactoside. The recombinant enzyme with a tag of six histidine residues at the C-terminus was purified 132-fold by affinity and gel-filtration chromatography. Analysis of the N-terminal amino acid sequence revealed that the first 42 amino acids were post-translationally cleaved off. The molecular mass of the purified LftM was approx. 54 kDa as determined by SDS/PAGE, which corresponded well with a predicted size from the nucleotide sequence of the lftM gene lacking 42 amino acids. The enzyme converted levan into difructose anhydride IV (DFA IV) with a K(m) of 2 mg/ml and a V(max) of 40.6 micromol/min at pH 7.0 and 40 degrees C. The pH-dependence study of the enzyme for DFA IV production showed that LftM had a broad pH optimum (5.0-8.0) and the pK(a) values obtained were 4.5 and 8.9 at 40 degrees C. These results suggest that the acidic residues at the active site may play important roles for the catalytic mechanism of the LFTase.
一个包含lftM基因的1.6 kb DNA片段被亚克隆到高表达载体pET - 29b中，该基因编码微杆菌属AL - 210的果聚糖蔗糖转移酶（LFTase），重组酶在大肠杆菌中过表达。用异丙基β - D -硫代半乳糖苷诱导后，大部分LFTase活性在细胞质部分被检测到。在C末端带有六个组氨酸残基标签的重组酶通过亲和层析和凝胶过滤层析纯化了132倍。对N末端氨基酸序列的分析表明，最初的42个氨基酸在翻译后被切除。通过SDS/PAGE测定，纯化后的LftM分子量约为54 kDa，这与lftM基因缺少42个氨基酸的核苷酸序列预测大小相符。该酶在pH 7.0和40℃条件下将果聚糖转化为二果糖酐IV（DFA IV），K(m)为2 mg/ml，V(max)为40.6 μmol/min。对该酶产生DFA IV的pH依赖性研究表明，LftM具有较宽的最适pH范围（5.0 - 8.0），在40℃下获得的pK(a)值分别为4.5和8.9。这些结果表明，活性位点的酸性残基可能在LFTase的催化机制中起重要作用。
