pThe thermostability of iStreptomyces lividans/i xylanase B (SlxB-cat) was significantly increased by the replacement of its iN/i-terminal region with the corresponding region from iThermomonospora fusca/i xylanase A (TfxA-cat) without observing a decrease in enzyme activity. In spite of the significant similarity between the amino acid sequences of the two xylanases, their thermostabilities are quite different. To facilitate an understanding of the contribution of structure to the thermostability observed, chimaeric enzymes were constructed by random gene shuffling and the thermostable chimaeric enzymes were selected for further study. A comparative study of the chimaeric and parental enzymes indicated that the iN/i-terminus of TfxA-cat contributed to the observed thermostability. However, too many substitutions decreased both the thermostability and the activity of the enzyme. The mutants with the most desirable characteristics, Stx15 and Stx18, exhibited significant thermostabilities at 70 °C with optimum temperatures which were 20 °C higher than that of SlxB-cat and equal to that of TfxA-cat. The ability of these two chimaeric enzymes to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes. These results suggest that these chimaeric enzymes inherit both their thermostability from TfxA-cat and their increased reactivity from SlxB-cat. Our study also demonstrates that random shuffling between a mesophilic enzyme and its thermophilic counterpart represents a facile approach for the improvement of the thermostability of a mesophilic enzyme./p
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机译：>青霉链霉菌木聚糖酶B（SlxB-cat）的热稳定性通过将其 N 末端区域替换为 Thermomonospora的相应区域而显着提高木聚糖酶A（TfxA-cat），但未观察到酶活性的降低。尽管两个木聚糖酶的氨基酸序列之间存在显着相似性，但它们的热稳定性却大不相同。为便于理解所观察到的结构对热稳定性的贡献，通过随机基因改组构建了嵌合酶，并选择了耐热的嵌合酶作进一步研究。对嵌合酶和亲本酶的比较研究表明，TfxA-cat的 N 端有助于观察到的热稳定性。然而，太多的取代降低了酶的热稳定性和活性。具有最理想特性的突变体Stx15和Stx18在70°C下表现出显着的热稳定性，最佳温度比SlxB-cat高20°C，与TfxA-cat相等。与亲本酶相比，这两种嵌合酶从木聚糖产生还原糖的能力得到增强。这些结果表明这些嵌合酶既继承了TfxA-cat的热稳定性，又继承了SlxB-cat的反应性。我们的研究还表明，嗜温酶及其嗜热对应物之间的随机改组是提高嗜温酶热稳定性的简便方法。
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