Yang R C, MacKenzie C R, Bilous D, Narang S A
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Appl Environ Microbiol. 1989 May;55(5):1192-5. doi: 10.1128/aem.55.5.1192-1195.1989.
A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.
将插入到pUC19中且编码内切木聚糖酶活性的环状芽孢杆菌基因组DNA的一个4.0千碱基（kb）片段进行了一系列亚克隆。发现一个1.0 kb的HindIII - HincII亚片段编码木聚糖酶活性。用一个在编码区上游含有额外0.3 kb序列的亚克隆观察到了最大表达水平。上游区域的增强子序列被认为是造成这些高表达水平的原因。Southern杂交分析表明，克隆的基因与枯草芽孢杆菌和多粘芽孢杆菌的基因组DNA杂交。携带克隆基因的大肠杆菌所表达的木聚糖酶活性主要位于细胞内部分。达到了高达7 U/ml或35 mg/升的水平。通过离子交换和凝胶渗透色谱法纯化了蛋白质产物。该木聚糖酶的分子量为20,500，等电点为9.0。
