Janer Carolina, Rohr Lukas M, Peláez Carmen, Laloi Maryse, Cleusix Valentine, Requena Teresa, Meile Leo
Department of Dairy Science and Technology, Instituto del Frío (CSIC), Ciudad Universitaria, Madrid, Spain.
Syst Appl Microbiol. 2004 May;27(3):279-85. doi: 10.1078/0723-2020-00274.
The ability of the beta-fructofuranosidase (EC 3.2.1.26) from Bifidobacterium lactis DSM 10140T to cleave a variety of fructooligosaccharides was characterised. We identified its gene on a cloned chromosomal DNA fragment by sequence similarity (69% identity) to the putative CscA protein encoded in the Bifidobacterium longum genome. The deduced amino acid sequence of 532 residues (59.4 kDa) appeared to be identical to the beta-fructofuranosidase from the same strain recently described by Ehrmann et al. (Curr. Microbiol. 2003, 46, 391-397). However, the characterisation of the heterologously expressed enzyme showed several discrepancies to the referred study. First, the B. lactis beta-fructofuranosidase gene was found to have 41% identity with CscA from E. coli in contrast to the 16% reported, therefore it was assigned to as CscA protein instead of BfrA. Second, we observed only low activity of the enzyme towards sucrose (6%) instead of the 100% previously reported. Instead, we measured highest activity (100%) of the enzyme with the oligofructose Raftilose as a substrate compared with the inulin of low degree of polymerisation Raftiline LS (29%) and the highly polymerised Raftiline HP (10%). Altogether, the enzyme showed high affinity to terminal beta(2-1) glycosyl linkages between fructose moieties. The Km values obtained for Raftilose, Raftiline LS and sucrose were 0.12, 7.08 and 8.37 mM, respectively, and V(max) values for the conversion to fructose were calculated to be 5, 21 and 17 micromol/min per mg of protein, respectively. Growth of B. lactis was supported by fructans of low degree of polymerisation (Raftilose and Raftiline LS), whereas we observed no growth with highly polymerised inulin (Raftiline HP).
对来自乳酸双歧杆菌DSM 10140T的β-呋喃果糖苷酶（EC 3.2.1.26）裂解多种低聚果糖的能力进行了表征。我们通过与长双歧杆菌基因组中编码的假定CscA蛋白的序列相似性（69%同一性）在克隆的染色体DNA片段上鉴定了其基因。推导的532个残基（59.4 kDa）的氨基酸序列似乎与Ehrmann等人最近描述的同一菌株的β-呋喃果糖苷酶相同（《当前微生物学》，2003年，46卷，391 - 397页）。然而，对异源表达酶的表征显示与上述研究存在一些差异。首先，发现乳酸双歧杆菌β-呋喃果糖苷酶基因与大肠杆菌的CscA有41%的同一性，而非报道的16%，因此将其归类为CscA蛋白而非BfrA。其次，我们观察到该酶对蔗糖的活性仅为6%，而非先前报道的100%。相反，与低聚合度菊粉Raftiline LS（29%）和高聚合度菊粉Raftiline HP（10%）相比，以低聚果糖Raftilose作为底物时，该酶的活性最高（100%）。总体而言，该酶对果糖部分之间的末端β(2 - 1)糖基键具有高亲和力。Raftilose、Raftiline LS和蔗糖的Km值分别为0.12、7.08和8.37 mM，转化为果糖的V(max)值经计算分别为每毫克蛋白质5、21和17微摩尔/分钟。低聚合度果聚糖（Raftilose和Raftiline LS）可支持乳酸双歧杆菌生长，而我们观察到高聚合度菊粉（Raftiline HP）无法支持其生长。
