Characterization of recombinant beta-fructofuranosidase from Bifidobacterium adolescentis G1.
Characterization of recombinant beta-fructofuranosidase from Bifidobacterium adolescentis G1.
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DOI：
10.1186/1752-153x-4-9
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发表时间：
2010-04-12
影响因子：
--
通讯作者：
Shiomi N
中科院分区：
文献类型：
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作者：
Omori T;Ueno K;Muramatsu K;Kikuchi M;Onodera S;Shiomi N
我们之前已经报道了青春型双歧杆菌β-呋喃果糖苷酶(β-FFase)的纯化和性质。该酶对低聚合度的低聚果糖表现出较高的水解酶活性。最近测定了长杆菌NCC2705和青春菌ATCC 15703的基因组序列，并预测了编码β-FFase的两个基因组序列中的CscA基因。本文介绍了青春型芽孢杆菌G1CscA基因的克隆、在大肠杆菌中的表达及其重组蛋白的性质。利用青春型双歧杆菌ATCC 15703的CscA基因信息，克隆了青春型双歧杆菌G1的CscA基因并进行了序列测定。纯化的青春型芽孢杆菌β-Fas酶N端氨基酸序列与青春型芽孢杆菌CscA基因推导的氨基酸序列完全一致。为了验证CSCA基因的翻译产物，将重组蛋白在大肠杆菌中进行了表达。经SDS-PAGE和MALDI-TOF-MS分析，纯化的重组酶的相对分子质量分别为66,000和60,300。该酶的最适pH为5.7，在pH 5.0~8.6范围内稳定。Km(Mm)、Vmax(μ摩尔/mg蛋白质/分钟)、k0(秒~(-1))和k0(mm~(-1)秒~(-1))和k0/Km(mm~(-1)秒-1)。分别为25.9、77、77.4、3.0。硝酸银、十二烷基硫酸钠和氯化汞对该酶的水解性有较强的抑制作用。重组酶具有与天然酶相似的特异性，对1-酮糖的亲和力较高，对蔗糖和菊粉的亲和力较低，但性质与天然酶略有不同。
We have previously reported on purification and characterization of β-fructofuranosidase (β-FFase) from Bifidobacterium adolescentis G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of B. longum NCC2705 and B. adolescentis ATCC 15703 were determined, and cscA gene in the both genome sequences encoding β-FFase was predicted. Here, cloning of cscA gene encoding putative β-FFase from B. adolescentis G1, its expression in E. coli and properties of the recombinant protein are described. Using the information of cscA gene from Bifidobacterium adolescentis ATCC 15703, cscA gene from B. adolescentis G1 was cloned and sequenced. The N-terminal amino acid sequence of purified β-FFase from B. adolescentis G1 was identical to the deduced amino acid sequences of cscA gene from B. adolescentis G1. To confirm the translated product of the cscA gene, the recombinant protein was expressed in Escherichia coli. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50°C. The Km (mM), Vmax (μmol/mg of protein/min), k0 (sec-1) and k0/Km(mM-1 sec-1) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO3, SDS, and HgCl2. The recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.