IDENTIFICATION AND CHARACTERIZATION OF CLUSTERED GENES FOR THERMOSTABLE XYLAN-DEGRADING ENZYMES, BETA-XYLOSIDASE AND XYLANASE, OF BACILLUS-STEAROTHERMOPHILUS-22
IDENTIFICATION AND CHARACTERIZATION OF CLUSTERED GENES FOR THERMOSTABLE XYLAN-DEGRADING ENZYMES, BETA-XYLOSIDASE AND XYLANASE, OF BACILLUS-STEAROTHERMOPHILUS-22
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DOI：
10.1128/aem.60.7.2252-2258.1994
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发表时间：
1994-07-01
影响因子：
4.4
通讯作者：
NANMORI, T
中科院分区：
文献类型：
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作者：
BABA, T;SHINKE, R;NANMORI, T
嗜热脂肪芽孢杆菌21是一种革兰氏阳性兼性嗜热需氧菌，可以利用木聚糖作为唯一的碳源。我们从土壤中分离到该菌株，纯化了其胞外木聚糖酶和β-木糖苷酶，并分析了这两种酶对木聚糖的两步降解（T。Nanmori，T.渡边河，巴西-地Shinke，A. Kohno和Y. Kawamura，J. Bacteriol. 172：6669-6672，1990）。分离到一株携带该细菌4.2kbp染色体片段的大肠杆菌Escherichia coli，作为重组质粒。它分泌活性β-木糖苷酶和木聚糖酶到培养基中。将该质粒导入对紫外线敏感的E. coliCSR 603，并对表达产物进行maxicell分析。携带β-木糖苷酶和木聚糖酶活性的蛋白质进行了鉴定，并估计其分子量由十二烷基硫酸钠-聚芳酰胺凝胶电泳分别为75和40 kDa。该值与从B的细胞制备的蛋白质的值相同。嗜热脂肪菌21.这两种酶的基因编码在一个3.4 kbp的PstI片段，来自4.2 kbp的染色体片段。相应地确定了4.2-kbp片段的核苷酸序列。β-木糖苷酶基因（xylA）位于木聚糖酶基因（xynA）的上游，具有可能的启动子和Shine-Dalgarno序列。后一个基因之前有两个可能的启动子，其位于前者的3 '末端编码区内的Shine-Dalgarno序列。因此，这两个基因似乎，至少部分，独立表达，这是在大肠杆菌中通过缺失分析实验证实。xynA基因属于纤维素酶-木聚糖酶家族F，具有6个在该家族中高度保守的区域。然而，β-木糖苷酶与PIR数据库（版本38.0）中的任何其他蛋白质均无可检测的相似性。
Bacillus stearothermophilus 21 is a gram-positive, facultative thermophilic aerobe that can utilize xylan as a sole source of carbon. We isolated this strain from soil, purified its extracellular xylanase and beta-xylosidase, and analyzed the two-step degradation of xylan by these enzymes (T. Nanmori, T. Watanabe, R. Shinke, A. Kohno, and Y. Kawamura, J. Bacteriol. 172:6669-6672, 1990). An Escherichia coli transformant carrying a 4.2-kbp chromosomal segment of this bacterium as a recombinant plasmid was isolated. It excreted active beta-xylosidase and xylanase into the culture medium. The plasmid was introduced into UV-sensitive E. coli CSR603, and its protein products were analyzed by the maxicell method. Proteins harboring beta-xylosidase and xylanase activities were identified, and their molecular masses were estimated by sodium dodecyl sulfate-polyarylamide gel electrophoresis to be 75 and 40 kDa, respectively. The values were identical to those of proteins prepared from cells of B. stearothermophilus 21. The genes for both enzymes were encoded in a 3.4-kbp PstI fragment derived from the 4.2-kbp chromosomal segment. The nucleotide sequence of the 4.2-kbp segment was accordingly determined. The beta-xylosidase gene (xylA) is located upstream of the xylanase gene (xynA) with a possible promoter and a Shine-Dalgarno sequence. The latter gene is preceded by two possible promoters acid a Shine-Dalgarno sequence that are located within the 3'-terminal coding region of the former. The two genes thus appear to be, at least partly, expressed independently, which was experimentally confirmed in E, coli by deletion analysis. The xynA gene was found to belong to cellulase-xylanase family F, possessing six regions that are highly conserved in this family. However, the beta-xylosidase has no detectable similarity to any other proteins in the PIR data base (release 38.0).