EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF AN EXO-BETA-D-FRUCTOSIDASE OF STREPTOCOCCUS-MUTANS
EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF AN EXO-BETA-D-FRUCTOSIDASE OF STREPTOCOCCUS-MUTANS
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DOI：
10.1128/jb.169.10.4507-4517.1987
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发表时间：
1987-10-01
影响因子：
3.2
通讯作者：
YASBIN, RE
中科院分区：
文献类型：
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作者：
BURNE, RA;SCHILLING, K;YASBIN, RE
利用大肠杆菌质粒载体构建变形链球菌GS-5基因文库，筛选能利用蔗糖作为唯一碳源和能源的细胞。重组质粒pFRU 1含有一个S.4.2-内切酶对插入片段。变形DNA，显示赋予这种表型。对pFRU 1编码的基因产物的进一步表征表明，该酶是β-对β-葡萄糖苷酶具有最高特异性的D-果糖苷酶。（2）6)-连接的果聚糖聚合物莱万。该酶还可以水解菊粉[β]。（2）1)-连接的果聚糖]、蔗糖和棉子糖，分别具有莱万所观察到的活性的34、21和12%。该基因（命名为fruA）在E.大肠杆菌中，这是通过β-葡聚糖酶的诱导或阻遏对基因产物活性缺乏影响来判断的。半乳糖苷酶启动子与克隆载体上的插入位点相邻。通过在变性和还原条件下在聚丙烯酰胺凝胶中对纯化的蛋白质进行银染色，从E.大肠杆菌（S. coli）培养上清中的蛋白质，以及S.在恒化器中以低稀释率用果糖作为唯一的碳水化合物源生长的变异株GS-5。这两种纯化的蛋白质在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中的表观分子量为140，000道尔顿，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中通过免疫印迹法与针对克隆基因产物的抗血清测定免疫相关和共迁移，并且在所有测试的物理和生化性质上相同。作用于果聚糖聚合物的酶的最适pH为5.5，在pH 4.0下保持显著量的活性。蔗糖降解的最适pH较宽且较低，峰值约为4.5。Hg ~（2+）和Ag ~（2+）几乎完全抑制酶活性，Cu ~（2+）部分抑制酶活性，F ~（2+）和Tris对酶活性无抑制作用，Mn ~（2+）和Co ~（2+）对酶活性有一定的促进作用。果聚糖聚合物被攻击外水解的酶，果糖是唯一的产品释放。在足够的时间内，莱万和菊粉都完全降解，没有产物抑制的证据。
A genetic library of Streptococcus mutans GS-5, constructed in an Escherichia coli plasmid vector, was screened for cells which could utilize sucrose as the sole carbon and energy source. The recombinant plasmid pFRU1, containing a 4.2-kilobase pair insert of S. mutans DNA, was shown to confer this phenotype. Further characterization of the gene product encoded by pFRU1 revealed that the enzyme was a .beta.-D-fructosidase with the highest specificity for the .beta.(2 .fwdarw. 6)-linked fructan polymer levan. The enzyme could also hydrolyze inulin [.beta.(2 .fwdarw. 1)-linked fructan], sucrose, and raffinose with 34, 21, and 12%, respectively, of the activity observed for levan. The gene (designated fruA) appeared to be expressed under its own control in E. coli, as judged by the lack of influence on gene product activity of induction or repression of the .beta.-galactosidase promoter adjacent to the insertion site on the cloning vector. The protein was purified to homogeneity, as judged by silver staining of purified protein in denaturing and reducing conditions in polyacrylamide gels, from sonic lysate of E. coli, as well as from culture supernatants of S. mutans GS-5 grown in a chemostat at low dilution rate with fructose as the sole carbohydrate source. Both purified proteins had an apparent molecular mass of 140,000 daltons in sodium dodecyl sulfate-polyacryalmide gel electrophoresis, were immunologically related and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as determined by Western blotting with antisera raised against the cloned gene product, and were identical in all physical and biochemical properties tested. The pH optimum of the enzyme acting on fructan polymers was 5.5, with a significant amount of activity remaining at pH 4.0. The optimum pH for sucrose degradation was broader and lower, with a peak at approximately 4.5. Enzyme activity was inhibited almost completely by Hg2+ and Ag2+, inhibited partially by Cu2+, not inhibited by fluoride ion or Tris, and slightly stimulated by Mn2+ and Co2+. Fructan polymers were attacked exohydrolytically by the enzyme, fructose being the only product released. With sufficient time, both levan and inulin were degraded to completion, with no evidence of product inhibition.