Poonperm Wayoon, Takata Goro, Okada Hiromi, Morimoto Kenji, Granström Tom Birger, Izumori Ken
Rare Sugar Research Center and Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki, Kagawa Prefecture 761-0795, Japan.
Appl Microbiol Biotechnol. 2007 Oct;76(6):1297-307. doi: 10.1007/s00253-007-1109-3. Epub 2007 Jul 25.
The L-rhamnose isomerase gene (L-rhi) encoding for L-rhamnose isomerase (L-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6xHis sequence at a C-terminal of the protein. The open reading frame of L-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced L-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant L-RhI exhibited maximum activity at 65 degrees C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60 degrees C for 60 min. The apparent affinity (K(m)) and catalytic efficiency (k(cat)/K(m)) for L-rhamnose (at 65 degrees C) were 4.89 mM and 8.36 x 10(5) M(-1) min(-1), respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50 degrees C, for D: -allose, L-mannose, D-ribulose, and L-talose from D-psicose, L-fructose, D-ribose and L-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant L-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.
来自兼性嗜热细菌苍白芽孢杆菌Y25的编码L-鼠李糖异构酶（L-RhI）的L-鼠李糖异构酶基因（L-rhi）被克隆，并在大肠杆菌中与蛋白质C端的6xHis序列协作进行了过表达。L-rhi的开放阅读框由1236个核苷酸组成，编码412个氨基酸残基，计算分子量为47,636 Da，与天然酶显示出良好的一致性。大量生产的L-RhI以可溶性蛋白的形式大量获得（470 mg/l肉汤）。重组酶通过使用Ni-NTA亲和柱色谱的一步纯化法纯化至同质。纯化的重组L-RhI在测定条件下于65℃（pH 7.0）表现出最大活性，而在60℃孵育60分钟后可保留90%的初始酶活性。L-鼠李糖（在65℃）的表观亲和力（K(m)）和催化效率（k(cat)/K(m)）分别为4.89 mM和8.36×10(5) M(-1) min(-1)。该酶分别与大肠杆菌和施氏假单胞菌相比，显示出相对较低水平的氨基酸序列相似性（42%和）、更高的热稳定性和不同的底物特异性。该酶在50℃对来自D-阿洛酮糖、L-果糖、D-核糖和L-塔格糖的D-阿洛糖、L-甘露糖、D-核酮糖和L-塔罗糖具有良好的催化活性，转化率分别为35%、25%、16%和10%，且无副产物污染。这些发现表明，来自苍白芽孢杆菌的重组L-RhI适合作为大规模生产稀有糖生产酶的新来源。
