Shendye A, Rao M
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
FEMS Microbiol Lett. 1993 Apr 15;108(3):297-302. doi: 10.1111/j.1574-6968.1993.tb06119.x.
A genomic DNA library of an alkaliphilic thermophilic Bacillus was constructed in Escherichia coli with pUC 8 vector and was screened using a Congo red xylan plate clearance assay. Six xylanase positive transformants having identical inserts showed immunological reactivity towards polyclonal antibodies raised against purified xylanase (M(r) 15,800) from the Bacillus. A 4.5-kb HindIII-EcoRI subfragment was found to code for two xylanases of M(r) 14,500 and 35,000. Equivalent amounts of xylanase activity were detected from IPTG induced and noninduced recombinants irrespective of the orientation of the 4.5-kb insert with respect to the lac promoter, indicating that xylanase gene expression was under the control of its own promoter. 95% of the xylanase activity (2 U/ml) was found in the extracellular culture filtrate. The hydrolysis of xylan by the recombinant xylanases yielded mainly xylobiose.
用pUC 8载体在大肠杆菌中构建了嗜碱嗜热芽孢杆菌的基因组DNA文库，并使用刚果红木聚糖平板清除试验进行筛选。六个具有相同插入片段的木聚糖酶阳性转化子对针对该芽孢杆菌纯化木聚糖酶（M(r) 15,800）产生的多克隆抗体表现出免疫反应性。发现一个4.5 kb的HindIII - EcoRI亚片段编码M(r) 14,500和35,000的两种木聚糖酶。无论4.5 kb插入片段相对于lac启动子的方向如何，IPTG诱导和未诱导的重组体中检测到的木聚糖酶活性量相当，这表明木聚糖酶基因表达受其自身启动子的控制。95%的木聚糖酶活性（2 U/ml）存在于细胞外培养滤液中。重组木聚糖酶对木聚糖的水解主要产生木二糖。
