Analysis of a glucanase gene from Bacteriodes oralis Ig4a

葡聚糖酶有清除牙菌斑的作用，可作为一种口服治疗药物。我们以前报道过从口腔菌群中分离的口腔拟杆菌Ig4a产生α -1,6-葡聚糖酶（葡聚糖酶）。本研究将口孢杆菌Ig4a葡聚糖酶基因克隆到大肠杆菌pUC8载体的HindIII位点，重组质粒命名为pGL1。pGL1含有一个来自B. oralis Ig4a的4.2千碱基对（kb） HindIII DNA片段的插入。含有pGL1的大肠杆菌产生的葡聚糖酶活性主要集中在细胞质中。对pGL1缺失和插入质粒的分析和DNA测序显示，该片段缺乏自己的启动子，并且一个长长的开放阅读框（ORF）从一端开始，在插入物的中间结束。ORF可编码一个82kd的蛋白。从该序列推断出的c -末端3氨基酸序列与从B. oral alis Ig4a中纯化的44 kd葡聚糖酶序列相匹配，结果证实了该序列的正确性，尽管该生物产生的葡聚糖酶分子量最大，为105 kd。以上结果表明，克隆的片段是葡聚糖酶基因的3'部分，并且在B. oral中，Ig4a葡聚糖酶是作为105 kd的蛋白种产生的，并被未知的蛋白酶加工成44 kd的蛋白种。

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.