Panbangred W, Kondo T, Negoro S, Shinmyo A, Okada H
Mol Gen Genet. 1983;192(3):335-41. doi: 10.1007/BF00392172.
The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and beta-xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and beta-xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and beta-xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the beta-xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not beta-xylosidase, was secreted into the medium in a B. pumilus culture.
短小芽孢杆菌IPO的7.7Mdal PstI片段含有木聚糖降解基因、木聚糖酶和β-木糖苷酶，将其插入pBR322的PstI位点并克隆到大肠杆菌C600中。由此形成的重组质粒命名为pOXN29。携带pOXN29的大肠杆菌中表达的木聚糖酶和β-木糖苷酶的量分别约为供体短小芽孢杆菌产生的活性的6%和20%。插入片段的反向定向分别使木聚糖酶和β-木糖苷酶的生产率提高了5倍和50倍。通过免疫学和化学标准表明，在大肠杆菌转化体中表达的两种酶与短小芽孢杆菌的酶没有区别。用BglII消化pOXN29产生两个片段；一个大小为6.7Mdal，包含整个pBR322和β-木糖苷酶基因，另一个为3.7Mdal，编码木聚糖酶。对转化体细胞中表达的酶的分析表明，两种酶都没有分泌到培养基、周质或膜结合物中，尽管在短小芽孢杆菌培养物中木聚糖酶分泌到了培养基中，但β-木糖苷酶没有。
