Analysis of a glucanase gene from Bacteriodes oralis Ig4a

葡聚糖酶已被报道可去除牙菌斑，可用作口服治疗剂。我们曾报道从口腔植物群中分离到一株口腔拟杆菌Ig 4a，该菌产α-1，6-葡聚糖酶（葡聚糖酶）。本研究克隆了B的葡聚糖酶基因。将口腔链球菌Ig 4a插入大肠杆菌pUC 8载体的HindIII位点，将获得的重组质粒命名为pGL 1。pGL 1含有来自B的4.2个内切酶对（kb）HindIII DNA片段的插入片段。口腔Ig 4a。E.大肠杆菌表达质粒pGL 1后，产生葡聚糖酶活性，酶活性主要定位于胞质。对pGL 1的缺失和插入质粒的分析和DNA测序表明，该片段缺乏自己的启动子，并且进一步发现一个长的开放阅读框（ORF）起始于插入片段的一端，终止于插入片段的中部。该开放阅读框可编码一个分子量为82 kd的蛋白质。从该核苷酸序列推导的C-末端3个氨基酸序列与从B纯化的44 kd葡聚糖酶的C-末端3个氨基酸序列相匹配的结果验证了该核苷酸序列。oralis Ig 4a，尽管该生物体产生具有最大分子量的105 kd种类的葡聚糖酶。以上结果共同表明，克隆片段是葡聚糖酶基因的3'部分，而在B中。口腔Ig 4a葡聚糖酶以105 kd蛋白质种类产生，并通过未知蛋白酶加工成44 kd种类。

Glucanases which have been reported to remove dental plaque could be used as an oral therapeutic agent. We previsously reported that Bacteroides oralis Ig4a isolated from oral flora, produced alpha-1,6-glucanase (dextranase). In the present work, we cloned a dextranase gene of B. oralis Ig4a into the HindIII site of pUC8 vector of Escherichia coli and the obtained recombinant plasmid was designated pGL1. pGL1 contained an insert of a 4.2 kilobase-pair(kb) HindIII DNA fragment derived from B. oralis Ig4a. E. coli containing pGL1 produced dextranase activity which was mainly localized in the cytoplasmic fration. Analyses of deletion and insertion plasmids of pGL1 and DNA sequencing revealed that the fragment lacked its own promoter and further that a long open reading frame (ORF) started at one of the ends and finished around the middle of the insert. The ORF could encode a protein of 82 kd. The nucleotide sequence was verified by the result that the C-terminal 3 amino acid sequence deduced from the nucleotide sequence matched with that of 44 kd dextranase purified from B. oralis Ig4a, although the organism produced a dextranase of 105 kd species with the largest molecular weight. The above results all together suggest that the cloned fragment is a 3' portion of the dextranase gene and that in B. oralis Ig4a dextranase was produced as a 105 kd protein species and processed to a 44 kd species by unknkown proteases.