Ryan Sinéad M, Fitzgerald Gerald F, van Sinderen Douwe
Alimentary Pharmabiotic Centre, National University of Ireland Cork, Western Road, Cork, Ireland.
Appl Environ Microbiol. 2005 Jul;71(7):3475-82. doi: 10.1128/AEM.71.7.3475-3482.2005.
An operon involved in fructooligosaccharide breakdown was identified in the genome of Bifidobacterium breve UCC2003. This 2.6-kb transcriptional unit was comprised of three genes that encoded a putative permease, a conserved hypothetical protein, and a beta-fructofuranosidase. Active transcription of the operon was observed when B. breve UCC2003 was grown on sucrose or Actilight, while transcription appeared to be repressed when the organism was grown on glucose, fructose, a combination of glucose and sucrose, or a combination of fructose and sucrose. The beta-fructofuranosidase encoded by this operon was purified and biochemically characterized. The optimum pH and temperature for catalytic activity were determined to be pH 6.0 and 37 degrees C, respectively, and there was a dependence on bivalent cations, particularly manganese. The Km and Vmax values for sucrose hydrolysis were determined to be 25 +/- 2 mM and 24 +/- 3 micromol min(-1) mg(-1), respectively. Interestingly, the enzyme was shown to specifically catalyze cleavage of the beta(2-1) glycosidic bond between glucose and its neighboring fructose moiety in sucrose and other fructooligosaccharides with a relatively low degree of polymerization, and there was no detectable activity towards the beta(2-1) glycosidic bond between two fructose moieties within the same substrate. To our knowledge, such an enzymatic activity has not previously been described in bifidobacteria or other gram-positive bacteria.
在短双歧杆菌UCC2003基因组中鉴定出一个参与低聚果糖分解的操纵子。这个2.6 kb的转录单元由三个基因组成，分别编码一个假定的通透酶、一个保守的假定蛋白和一个β-果糖呋喃糖苷酶。当短双歧杆菌UCC2003在蔗糖或Actilight上生长时，观察到该操纵子的活跃转录，而当该菌在葡萄糖、果糖、葡萄糖和蔗糖的组合或果糖和蔗糖的组合上生长时，转录似乎受到抑制。对该操纵子编码的β-果糖呋喃糖苷酶进行了纯化和生化特性分析。确定其催化活性的最适pH和温度分别为pH 6.0和37℃，并且依赖于二价阳离子，尤其是锰。蔗糖水解的Km和Vmax值分别确定为25±2 mM和24±3 μmol min-1 mg-1。有趣的是，该酶显示出特异性催化蔗糖和其他聚合度相对较低的低聚果糖中葡萄糖与其相邻果糖部分之间的β(2-1)糖苷键的裂解，并且对同一底物中两个果糖部分之间的β(2-1)糖苷键没有可检测到的活性。据我们所知，这种酶活性以前在双歧杆菌或其他革兰氏阳性菌中尚未有过描述。
