Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced β-glucanase and xylanase expressed in Escherichia coli
Construction and characterization of a bifunctional fusion enzyme of Bacillus-sourced β-glucanase and xylanase expressed in Escherichia coli
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DOI：
10.1111/j.1574-6968.2006.00367.x
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发表时间：
2006-08-01
影响因子：
2.1
通讯作者：
Hu, Chun-Xia
中科院分区：
文献类型：
--
作者：
Lu, Ping;Feng, Ming-Guang;Hu, Chun-Xia
通过端到端融合的方法构建了淀粉液化芽孢杆菌葡聚糖酶(Glu，24.4 kDa)和枯草杆菌木聚糖酶(Xyl，21.2 kDa)的嵌合基因Glu-Xyl，并在大肠杆菌中成功表达。纯化的融合蛋白大小为46.1 kDa，具有葡聚糖酶和木聚糖酶活性。与亲本酶相比，Glu部分的K-m降低(0.66倍)，K-cat增加(2.75倍)，而Xyl部分的K-m增加(1.37倍)，K-CAT降低(0.79倍)。这表明Glu和Xyl部分的催化效率(K-cat/K-m)下降了31%，净增加了3.15倍。在40-90℃或pH 3.0-10.0范围内，比较了这两个部分与亲本酶的活性和稳定性。尽管有一些变化，但谷氨酸部分和母体的最适温度为40℃，pH为9.0，而木糖的最适温度为50-60℃，pH为9.0。因此，融合酶Glu-Xyl是双功能的，与木聚糖酶活性降低相关的葡聚糖酶活性显著增强。
A chimeric gene, Glu-Xyl, encoding Bacillus amyloliquefaciens glucanase (Glu, 24.4 kDa) and Bacillus subtilis xylanase (Xyl, 21.2 kDa), was constructed via end-to-end fusion and expressed successfully in Escherichia coli. The purified fusion protein (46.1 kDa) exhibited both glucanase and xylanase activities. Compared with parental enzymes, the Glu moiety was characterized by kinetic parameters of decreased K-m (0.66-fold) and increased K-cat (2.75-fold), whereas the Xyl moiety had an increased K-m (1.37-fold) and decreased K-cat (0.79-fold). These indicate a 3.15-fold net increase and a 31% decrease in catalytic efficiency (K-cat/K-m) of the Glu and Xyl moieties. Activities and stabilities of both moieties at 40-90 degrees C or pH 3.0-10.0 were compared with those of the parental enzymes. Despite some variations, common optima were 40 degrees C and pH 9.0 for the Glu moiety and parent, and 50-60 degrees C and pH 9.0 for the Xyl counterparts. Thus, the fusion enzyme Glu-Xyl was bifunctional, with greatly enhanced glucanase activity associated with a decrease in xylanase activity.