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首页> 外文期刊>Applied and Environmental Microbiology >Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli.
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Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli.

机译：拟杆菌琥珀酸杆菌木聚糖酶基因的分子克隆及其在大肠杆菌中的表达。

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A gene coding for xylanase synthesis in Bacteroides succinogenes was isolated by cloning, with Escherichia coli HB101 as the host. After partial digestion of B. succinogenes DNA with Sau3A, fragments were ligated into the BamHI site of pBR322 and transformed into E. coli HB101. Of 14,000 colonies screened, 4 produced clear halos on Remazol brilliant blue-xylan agar. Plasmids from two stable clones recovered exhibited identical restriction enzyme patterns, with the same 9.4-kilobase-pair (kbp) insert. The plasmid was designated pBX1. After subcloning of restriction enzyme fragments, a 3-kbp fragment was found to code for xylanase activity in either orientation when inserted into pUC18 and pUC19. The original clone possessed approximately 10-fold higher xylanase activity than did clones harboring the 3-kbp insert in pUC18, pUC19, or pBR322. The enzyme was partially secreted into the periplasmic space of E. coli. The periplasmic enzyme of the BX1 clone had 2% of the activity on carboxymethyl cellulose and less than 0.2% of the activity on p-nitrophenyl xyloside and a range of other substrates that it exhibited on xylan. The xylanase gene was not subject to catabolite repression by glucose or induction by either xylan or xylose. The xylanase activity migrated as a single broad band on nondenaturing polyacrylamide gels. The Km of the pBX1-encoded enzyme was 0.22% (wt/vol) of xylan, which was similar to that for the xylanase activity in an extracellular enzyme preparation from B. succinogenes. Based on these data it appears that the xylanase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to the B. succinogenes enzyme(s).
机译：以大肠杆菌HB101为宿主，通过克隆分离出琥珀酸琥珀酸杆菌中编码木聚糖酶合成的基因。用Sau3A部分消化琥珀酸芽孢杆菌DNA后，将片段连接到pBR322的BamHI位点并转化到大肠杆菌HB101中。在筛选的14,000个菌落中，有4个在Remazol明亮的蓝木聚糖琼脂上产生了清晰的光晕。从两个稳定的克隆中回收的质粒显示出相同的限制性酶图谱，具有相同的9.4碱基对（kbp）插入片段。该质粒命名为pBX1。将限制酶片段亚克隆后，发现一个3 kbp片段在插入pUC18和pUC19时可在任一方向编码木聚糖酶活性。原始克隆的木聚糖酶活性比在pUC18，pUC19或pBR322中带有3-kbp插入片段的木聚糖酶活性高约10倍。该酶部分分泌到大肠杆菌的周质空间中。 BX1克隆的周质酶对羧甲基纤维素的活性为2％，对对硝基苯基木糖苷及其一系列在木聚糖上表现出的其他底物的活性小于0.2％。木聚糖酶基因不受葡萄糖的分解代谢物抑制或木聚糖或木糖的诱导。木聚糖酶活性在非变性聚丙烯酰胺凝胶上以单个宽带迁移。 pBX1编码的酶的Km为木聚糖的0.22％（wt / vol），与来自琥珀酸芽孢杆菌的细胞外酶制剂中的木聚糖酶活性相似。基于这些数据，似乎在大肠杆菌中表达的木聚糖酶基因是完全功能性的，并且编码具有与琥珀酸芽孢杆菌的酶相似的性质的酶。

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